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Regulation of protein kinases during postnatal development of rat heart Kim, Sung Ouk 1998

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REGULATION OF PROTEIN KINASES DURING POSTNATAL DEVELOPMENT OF RAT HEART by SUNG OUK KIM B. Sc. (Pharra), University of Manitoba, 1992 A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Faculty of Graduate Studies, Division of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard University of British Columbia October, 1998 © Sung Kim, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ' f W l f M ffiUrb\Ul .Cf jV.-A.ft> A The University of British Columbia Vancouver, Canada Date nd- 1^ , l ^ f t DE-6 (2/88) ABSTRACT The loss of ability to proliferate, the shift in the energy dependency from carbohydrate to fatty acids and the reduction in capability to resist ischemia are some of the key phenomena observed during postnatal development of the heart. These distinct phenotypic changes can be attributed to the regulation of intracellular signal transduction proteins. To investigate how these protein kinases are expressed during postnatal development, 44 different protein kinases were studied by Western blotting analysis. Among them protein kinases, implicated in cell proliferation and cell cycle including protein kinase C, cyclin-dependent protein kinases and mitogenic M A P kinases, declined during the postnatal development. The specific activities of E r k l and Erk2 declined with age, which was concurrent to the decrease of C D K 1 activity. The protein kinases involved in stress-responses such as M l k 3 , M e k k l , S e k l , M k k 3 and Mapkapk2, increased in expression during postnatal development. The proto-oncogene-encoded kinases Mos and Tpl2 were up-regulated. The expression of the insulin-activated kinases (PI3K, P K B , S 6 K and C K 2 ) was down-regulated by 40-60% with age. The activities of P K B , S 6 K and C K 2 were also decreased with age. Tissue distribution studies of adult rats revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in the heart, whereas the down-regulated protein kinases were generally expressed in the heart at relatively lower amounts than in most other tissues. To investigate the signalling pathways of these kinases, isolated cardiomyocytes or isolated whole hearts were treated with various agonists. These results indicate that E r k l can be regulated via P K C but not by c A P K nor c G P K , whereas Mapkapk2 can be controlled by c G P K ii but not by the other two kinases. E r k l and Erk2 were also activated by endothelin-1 and adenosine in isolated cardiomyocytes. Insulin led to activation of P K B , S 6 K and C K 2 , whereas E r k l and Erk2 were not stimulated. To investigate novel protein kinases which undergo developmental changes, the extracts of rat heart ventricles were incubated in the presence of [ y - 3 2 P ] A T P and then the phosphoproteins were resolved by using S D S - P A G E . This assay revealed at least 8 phosphoproteins that underwent change during development. Among them, a 40-kDa phosphoprotein appeared to be an autophosphorylating protein-histidine kinase, which may represent the first of its kind detected in a mammalian system. iii TABLE OF CONTENTS Page Abstract ii Table of contents iv List of tables x List of figures xi List of abbreviations xv Acknowledgments xxii Dedication xxiii 1. Introduction 1 1.1 Definition of postnatal development 1 1.2. Overview of heart development 3 1.2.1. Physical and structural aspects of postnatal development of the heart. 4 1.2.2. Molecular studies on the mechanism of cardiac myocyte terminal differentiation 7 1.3. Three paradigms for protein kinase signal transduction mechanisms and their roles in postnatal development of the heart 12 1.3.1. The second messenger- and cyclin-dependent protein kinases (CDKs) in proliferation and differentiation of cardiomyocytes 16 1.3.2. Mitogenic protein kinase ( M A P K ) signalling pathways in cell proliferation/differentiation and ischemia of cardiomyocytes 19 1.3.3. Activation of M A P kinases by cardiac ischemia/reperfusion and its role in cardiac preconditioning 28 1.3.4. Regulation of myocardial carbohydrate metabolism during postnatal development and ischemia 31 1.3.5. Insulin mediated signalling pathways in the regulation of myocardial carbohydrate metabolism and protein synthesis 37 1.3.6. Histidine protein kinase and the two-component regulatory system 39 1.3.6.1. Chemical characteristics of phosphohistidine 40 iv 1.3.6.2. The two-component regulatory system in prokaryotes 41 1.3.6.3. Lower eukaryotic two-component regulatory system in Hog 1 M A P kinase pathway 44 1.3.6.4. Histidine kinases in mammalian systems 46 2. Hypothesis 48 3. Objectives 49 4. Methods and materials 50 4.1. Materials 50 4.2. Animals 54 4.3. Methods 54 4.3.1. Preparation of various rat tissues 54 4.3.2. Preparation of isolated rat ventricular myocytes 55 4.3.3. Stimulation of ventricular tissues and cardiomyocytes 56 4.3.3.1. In vivo treatment of insulin by tail vein injection 56 4.3.3.2. Preparation in Langendorff-perfused isolated rat hearts 56 4.3.3.3. Treatment of isolated adult ventricular myocytes with various agents 57 4.3.4. Determination of protein concentration 57 4.3.5. Column chromatography 57 4.3.5.1. Ion-exchange column chromatography 57 4.3.5.2. Superose 12 gel-filtration chromatography 58 4.3.6. SDS-polyacrylamide gel electrophoresis, transblotting and immunoblotting 58 4.3.7. Stripping of Western blot membrane 60 4.3.8. Two-dimensional electrophoresis 60 4.3.9. Ge l and membrane staining 61 4.3.9.1. Ponceau staining 61 4.3.9.2. Amido black staining 61 4.3.9.3. Silver staining 61 4.3.9.4. Colloidal silver staining for membranes 61 V 4.3.10. Autoradiography and development of film 62 4.3.11 Protein kinase assays 62 4.3.11.1. Membrane of M B P , c-Jun and hsp27 phosphotransferase activities from crude extracts 62 4.3.11.2. Measurement of M B P , c-Jun, hsp27, S6-10, casein phosphotransferase activities from column fractionated extracts 63 4.3.11.3. Immunoprecipitation and determination of protein kinase activities 64 4.3.11.4. In situ M B P kinase assay: "in-gel" kinase assay 64 4.3.11.5. In vitro protein phosphorylation and autophosphorylation 64 4.3.11.6. Photoaffinity labeling of P K X with azido-ATP 65 4.3.12. Phosphoamino acid analysis 65 4.3.12.1. Basic phosphoamino acid analysis using thin layer chromatography 65 4.3.12.2. Basic phosphoamino acid analysis using Mono Q column 66 4.3.13. Densitometry and statistical analysis 66 4.4. Purification of 40-kDa histidine kinase from bovine heart ventricle 67 4.4.1. Homogenization of bovine heart ventricle 67 4.4.2. Ammonium sulfate precipitation of crude extracts 67 4.4.3. Desalting by G25 Sephadex filtration 67 4.4.4. Negative purification by Q-Sepharose and DEAE-cel lulose column chromatography 68 4.4.5. SP-Sepharose column chromatography 68 5. Results 69 5.1. Second messenger-and cyclin-dependent protein kinases 69 5.1.1. Expression of cyclic-nucleotide dependent protein kinases: c A P K and c G P K 69 5.1.2. Expression of Ca 2 + / l i p id - and calmodulin-dependent protein kinases: P K C and C a M P K I I 74 5.1.3. Expression of cyclin-dependent protein kinases (CDKs) 79 5.1.4. Expression of C D K regulating kinases: W E E 1 and C D K 7 85 5.1.5. Developmental regulation of C D K 1 activity 88 VI 5.2. Mitogen-activated protein kinase pathways during postnatal development of the rat heart 92 5.2.1. Expression of M A P kinase kinase kinase ( M A P K K K s ) involved in the regulation of E r k l and Erk2: Raf, Mos and Cot 92 5.2.2. Expression of M A P kinase kinase kinases ( M A P K K K s ) involved in the regulation of J N K and p38 kinase: Paks, M l k 3 and T a k l 98 5.2.3. Expression of M A P kinase kinases ( M A P K K s ) , M A P kinases ( M A P K s ) and their substrate kinases 106 5.2.3.1. Expression of E r k l and Erk2, and their up- and downstream kinases 106 5.2.3.2. Expression of J N K s and direct upstream kinases 109 5.2.3.3. Expression of p38 and its direct up- and downstream kinases 113 5.2.4. M B P , GSP-c-Jun, and heat shock protein 27 (hsp27) phosphotransferase activities during the postnatal development of rat heart ventricles 118 5.2.5. Isoform specific activities of Raf, Erk and Rsk during the postnatal development of rat heart ventricles 122 5.3. Regulation of M A P kinase signalling cascades in the heart and their role in ischemia preconditioning 128 5.3.1. Regulation of M A P kinases by cyclic-nucleotide dependent protein kinases and P K C 128 5.3.2. Nitric oxide mediated activation of E r k l , Erk2 and p38 M A P kinases in isolated rat cardiomyocytes 131 5.3.3. Regulation of E r k l by endothelin-1 and adenosine in perfused whole hearts and isolated cardio myocytes of rat heart 134 5.3.4. Activation of Mapkapk2 by adenosine and other stimuli 137 5.4. Insulin regulated protein kinases during postnatal development of rat heart 144 5.4.1. In vivo insulin activation of protein-serine kinases in rat heart 144 5.4.2. Developmental regulation of the P I 3 K / P K B / S 6 K pathway in rat heart 147 5.4.3. Developmental regulation of G S K 3 and C K 2 in rat heart ventricles 157 vii 5.5. Phosphoproteins involved in the postnatal development of rat heart ventricles 166 5.5.1. Detection and characterization of endogenous phosphoprotein of ventricular muscle and ventricular myocytes extracts during development of rat heart 166 5.5.2. Identification and characterization of phosphoproteins in Resource Q fractionated extracts of rat heart ventricles 172 5.5.3. Biochemical characterization and tissue distribution of the 40-kDa protein in rat 176 5.5.3.1. Preliminary biochemical characterization 176 5.5.3.2. Gel-filtration assays to investigate the protein complex 179 5.5.3.3. Confirmation that the 40-kDa protein is a protein kinase 183 5.5.3.4. Phosphoamino acid analysis of the 40-kDa protein kinase 183 5.5.3.5. Tissue distribution of 40-kDa histidine protein kinase in adult rat tissues 188 5.5.4. Purification of a mammalian 40-kDa histidine protein kinase from bovine heart ventricle ^gg 6. Discussion 193 6.1. Expression of cyclic nucleotide-dependent protein kinases and their role in the regulation of M A P kinases 193 6.2. Expression of protein kinase C and calmodulin-dependent protein kinase-2 196 6.3. Developmental expression of C D K s and regulation of C D K 1 activity 197 6.4. M A P kinases signalling cascades during the development of rat heart 202 6.5. Insulin signalling cascades during development of rat heart 209 6.6. Regulation of M A P kinases by various stimuli 210 6.7. Regulation of M A P kinases by adenosine and its role in ischemic preconditioning of the heart 213 6.8. Phosphoprotein during development of rat heart ventricle and identification of a novel histidine protein kinase 217 7. Summary and conclusions 221 Future studies 224 Bibliography 224 Appendix I 268 ix LIST OF TABLES 1 . List of antibodies and amino acid sequences of synthetic peptides used to raise antibodies 2. Purification of the 40-kDa histidine kinase form bovine heart LIST OF FIGURES Page 1. Approximate profile for selected developmental changes in the small 2 mammalian heart 2. Three paradigms for signal transduction mechanism in prokaryotes and eukaryotes 13 3. Putative signalling pathways in the activation of M A P kinases by seven transmembrane domain receptors 21 4. Putative role of insulin and other signalling pathways in the regulation of carbohydrate metabolism in the heart 33 5. Expression of cyclic-nucleotide dependent protein kinases during postnatal development of rat ventricle 70 6. Subcellular expression of second messenger- and cyclin dependent-protein kinases in 1-day old ventricles and isolated ventricular myocytes 72 7. Expression of P K C and C a M P K - 2 during postnatal development of rat ventricle 75 8 Expression of cyclin-dependent kinases during postnatal development of rat ventricle 80 9 Expression of C D K 8 and Kkialre during postnatal development of rat ventricle 83 10 Expression of C D K 7 and Wee l during postnatal development of rat ventricle 86 11 Activity of C D K 1 during postnatal development of rat ventricle 89 12 Expression of second messenger and cyclin-dependent kinases during the postnatal development of rat heart (1-365 day time period) 90 13 Expression of M A P kinase kinase kinase ( M K K K s ) involved in the regulation of E r k l and Erk2 during postnatal development of rat ventricle 93 14 Subcellular expression of protein kinases involved in M A P kinase pathways in isolated ventricular myocytes 97 15 Expression of p21-activated kinases during postnatal development of rat ventricle 100 xi 16 Expression of M A P kinase kinase kinase ( M K K K s ) involved in the regulation of I N K and p38 during postnatal development of rat ventricle 104 17 Expression of E r k l and Erk2 and their up- and downstream kinases 107 18 Expression of S A P K and the direct upstream kinase Sek l 111 19 Expression of p38 Hog and its direct up- and downstream kinases 114 20 Expression of M A P kinase signal cascades in postnatal developing rat heart (1-365 day time period) 117 21 Phosphotransferase activities toward M B P , GST-c-Jun, and heat shock protein 27 (hsp27) during postnatal development of rat ventricle 119 22 M B P phosphotransferase activities of Resource Q fractionated extracts during postnatal development of rat ventricle 121 23 c-Jun and hsp27 phosphotransferase activities of Resource Q fractionated extracts during postnatal development of rat ventricle 123 24 The total and specific activities of Raf l and RafA, E r k l and Erk2, and R s k l and Rsk2 during postnatal development of rat ventricle 125 25 Regulation of M A P kinases mediated by cyclic-nucleotides and P K C in isolated adult rat cardiomyocytes 129 26 C y c l i c - G M P dependent- and independent-activation of M A P kinases by S N P in isolated adult rat cardiomyocytes 132 27 Activation of E r k l by endothelin 1 in isolated adult rat ventricular myocytes 135 28 Activation of E r k l and Erk2 by adenosine in isolated perfusing rat heart and isolated cardiomyocytes 138 29 Activation of Mapkapk2 by adenosine in isolated perfusing rat heart ventricle and isolated adult rat cardiomyocytes 140 30 Regulation of Mapkapk2 by anisomycin and sorbitol 143 31 Activation of protein kinase B (PKB) and p70 S6 kinase (S6K) by insulin in rat ventricle 145 32 Activation of casein kinase 2 (CK2) by insulin in rat ventricle 148 xii 33 Lack of activation of M A P kinases E r k l and Erk2 by insulin in rat ventricle 150 34 Expression of PI3K during postnatal development of rat ventricle 153 35 Subcellular expression of insulin-regulated protein kinases in isolated ventricular myocytes 154 36 Expression and activity of P K B during postnatal development of rat ventricle 155 37 Expression and activity of p70 S 6 K during postnatal development of rat ventricle 158 38 Expression of G S K 3 during postnatal development of rat ventricle 160 39 Basal activity of casein kinase 2 (CK2) during postnatal development of rat ventricle 162 40 Expression of p70 S6 kinase signal cascades in postnatal developing rat heart 165 41 Identification of phosphoproteins during postnatal development of rat ventricle 167 42 Effects of various inhibitors in in vivo phosphorylation and the stability of phosphoproteins in boiling 169 43 Identification and characterization of phosphoproteins in isolated ventricular myocytes 173 44 In vitro phosphorylatoion of endogenous proteins in Resource Q fractionated extracts during development of rat ventricle 174 45 Preliminary biochemical characterization of 40-kDa phosphoprotein from adult rat ventricles 177 46 Effects of ethanol and geninstein in the partially purified 40-kDa protein histidine kinase in the adult rat ventricle 178 47 Effects of divalent cations in the autophosphorylation of 40-kDa histidine protein kinase 180 48 Time course of 40-kDa kinase autophosphorylation reaction 181 xiii 49 Partial purification of 40-kDa histidine protein kinase by F P L C gel filtration 182 50 A z i d o - A T P photolabelling of 40-kDa histidine protein kinase 184 51 Phosphoamino analysis of the 40-kDa histidine protein kinase in rat heart 186 ventricle 52 Tissue distribution of 40-kDa histidine protein kinase in Resource Q fractionated adult rat 190 xiv LIST OF ABBREVIATIONS 8-Br-cGMP 8-Br-cyclic G M P 3 2 P phosphorous-32 p, micro (1 x 10"6) A l adenosine receptor A l receptor aa amino acid A C C acetyl C o A carboxylase A d adipose A D P adenosine 5'-diphosphate A L P alkaline phosphatase A M P adenosine 5'-monophosphate A M P K AMP-dependent protein kinase A N G I I angiotensin II A P S ammonium persulfate A T P adenosine 5'-triphosphate A z i d o - A T P 8-azido-adenosine-5' -triphosphate BIS N,N'-methylene-bis-acrylamide B C I F 5-bromo-4-chloro-indoly phosphate B C I P 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt B S A bovine serum albumin Br brain C-terminal carboxyl-terminal C a 2 + free calcium ions C a M P K Ca 2 +/calmodulin-dependent protein kinase XV c A M P adenosine 3':5'-cyclic monophosphate c A P K cAMP-dependent protein kinase C D K cyclin-dependent protein kinase c G M P guanosine 3':5'-cyclic monophosphate c G P K cGMP-dependent protein kinase C i Curie, 2.22 x 10 1 2 disintegrations per minute C K casein kinase Cot cancer Osaka thyroid cpm counts per minute C P T carnitine-parmitoyl transferase C T carboxyl terminus, carboxyl terminal D A G diacylglycerol d H 2 0 distilled, deionized water D E A E diethylaminoethyl D M S O dimethylsulfoxide D T T dithiothreitol E C L enhanced chemiluminescence E D T A ethylene diamine tetraacetic disodium salt E G T A ethylene glycol bis(P-aminoethylether)-N,N,N'N'-tetraacetic acid F P L C fast protein liquid chromatography Erk extracellular signal-regulated kinases ET-1 endothelin-1 Fsk forskolin g gravitational force xvi g gram G E F guanidine nucleotide exchange factor gens genistein G L U T glucose transporter Grb2 growth factor receptor binding protein-2 GS glycogen synthase G T P guanidine 5'-triphosphate G P glycogen phosphorylase G S K glycogen synthase kinase G S T glutathione S-transferase h hour H E P E S N-(2-hydroxyethyl)piperazine-N'(2-ethanesulfonic acid) Herbm herbimycin HH1 histone H I H K hexokinase-II H O G high osmolarity glycerol H P L C high pressure liquid chromatography Hsp27 heat shock protein 27 Ht heart IEF isoelectric focusing Ig immunoglobulin In intestine IP 3 inositol-1,4,5-trisphosphate ip immunoprecipitation xvii IPG immobilized pH gradient IRS-1 insulin receptor substrate-1 I U international units I .V. intravenous J N K c-Jun-N terminal kinase K d kidney kDa kilodalton K K Kkialre 1 litre L u lung L v liver M mole/litre m A milliamp M A P K mitogen-activated protein kinase Mapkapk2 mitogen-activated protein kinase activated protein kinase-2 M B P myelin basic protein 2 - M E 2-mercaptoethanol M E K M A P K / E r k kinase M E K K M E K kinase M E M Jolik-modified minimum essential medium mg milligram min minute ml millilitre M l k mixed lineage kinase xviii M k k M A P kinase kinase Mnk M A P kinase signal-integrating kinase mol mole M O P S 3-(N-morpholino)propanesulfonic acid M r relative molecular mass n number of separate experiments N2 nitrogen N B T nitro blue tetrazolium n M nanomolar NP-40 Nonidet P-40 N T N-terminus or NH2-terminus 0 2 oxygen O D Q lH-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one P A G E polyacrylamide gel electrophoresis P A K p21-activated kinase P B S phosphate-buffered saline P F K 6-phosphofructo-l kinase P C preconditioning, preconditioned P D K phosphatidylinositol 3,4-biphosphate-dependent kinase p H negative logarithm of the hydrogen ion concentration P H Pleckstrin homology P i inorganic phosphate (PO4 3 ) PI3K phosphatidylinositol 3-kinase P K B protein kinase B xix P K C Ca +/phospholipid-dependent protein kinase P K I P K A inhibitor peptide P M A phorbol 12-myristate 13-acetate P M S F phenylmethylsulfonyl fluoride PP-1 protein phosphatase-1 P R A K p38-regulated/activated protein kinase PtdIns(3,4,5)P3 phosphatidylinositol-(3,4,5)-phosphate P V D F polyvinylidene difluoride Pyrd pyridine Rb retinoblastoma rpm revolution per minute R K reactivating kinase R S K ribosomal S6 kinase R T K receptor tyrosine kinase s second S6-10 ribosomal S6 peptide, R R L S S L R A S A P K stress-activated protein kinase S B T I soybean trypsin inhibitor S.D. standard deviation SDS sodium dodecyl sulfate S D S - P A G E SDS-polyacrylamide gel electrophoresis Sek stress-activated protein kinase kinase S H Src homology Skm skeletal muscle XX S N P sodium nitroprusside Sp spleen SPT 8-(P-sulfophenyl)theophylline S T M R seven transmembrane domain receptor Tak TGF(3-activated protein kinase T B S Tris-buffered saline T B S T T B S containing Tween 20 (0.2%, w/v) T E M E D N,N,N'N'-tetramethylethylenediamine Th thymus T L C thin-layer chromatography Tpl-2 tumor progression locus 2 TRIS tris(hydroxymethyl)aminomethane Ts testis U D P G uridine diphosphate glucose uv ultraviolet V o l volume X times A C K N O W L E D G E M E N T S I would like to express my sincere gratitude to my supervisors, Dr. Sidney Katz and Dr. Steve Pelech for their guidance and support throughout this study. I would also like to thank the members of my research committee: Dr. Roger Brownsey, Dr. Jack Diamond, Dr. Robert Harris and Dr. Marc Levine. I would like to acknowledge the contributions of Kinetek Pharmaceuticals Inc. for providing the various antibodies for Western blots. In addition, I wish to acknowledge Dr. H.R. Matthew, University of California at Davis, for performing alkaline phosphoamino acid analysis. I am appreciative of my past and present colleagues Chris Siow, Jasbinder Sanghera, Diana Lefebvre, Brooke Koide, Chrystal Palaty, X i o Hao, Sharon Johnstone, Ling Wu , Baljinder Salh, David Charest, Donna Morrison, Lorin Charlton, Yan Jun X u , Ravenska Wagey, Jing-Sung Tao, Tony Marotta, Mehrnaz Izagnegahdar, Mohammed Sayed, Jadvinder Dhesi, Vanessa Nahachewsky, Peggy Irwin and Christopher Tudan, for their constant support and critical discussion. I would also like to especially thank to M r . Mohammed Hasham for his invaluable help on the laboratory bench as well as in the computer programs. I also wish to thank Maggie Hampong, Harry Paddon, Georgia Tai and Catherine Sutter for their technical and administrative supports. I would like to acknowledge the financial support form the Medical Research Council of Canada for awarding me a five year Studentship to pursue my graduate research. Lastly I wish to appreciate my family members for their commitment, sacrifice, understanding and unconditional support. xxii With love To my family Geun-Ju, Bernard and Annabelle xxiii 1. INTRODUCTION 1.1 Definition of postnatal development. For many decades, the biology of the developing heart has been studied and the structure, function and metabolism of neonatal hearts have been discovered to be different from those of the adult hearts. The postnatal development from "immature" to "mature" or "neonate" to "adult" is a process of natural progression of the heart. There have been no commonly agreed definitions for "neonate" or "immature". The great difficulties in defining the time frame for the postnatal development result from the variations in the rates and sequences which change from tissues to tissues, organs to organs and species to species, and which may depend on the index under consideration. Moreover, even the adult stage is not stable and continuously changes in structure and function associated with physiological and pathophysiological processes. In the rat, most of the adult characteristics of the heart are manifested by 30 to 50 days after birth. During this postnatal period the heart undergoes dramatic changes in morphological, mechanical, electrophysiological and biochemical parameters (Rakusan, 1984; Zak, 1984b; Riva and Hearse, 1991; Michalak, 1987; Rumyantsev, 1996). Figure 1 (obtained from Riva and Hearse, 1991) graphically depicts a generalized overview of the relationship between various developmental changes and age in rat heart. A t the age of 30-35 days, most adult characteristics, such as blood pressure, heart rate, myocardial sodium content, calcium paradox, oxygen consumption, and resistance to ischemia, are observed. However, others, such as myocardial glycogen content and body weight, are still under progression even after this period. In the present study, I will , for the sake of consistency, use the term "postnatal development" when rats are between the birth to 50 days old and "adult" when rats are 50 to 365 days old. AGE (days) Figure 1. Approximate profile for selected development changes in the rat/mouse heart (obtained from Riva and Hearse, 1991). 2 1.2. Overview of heart development. The development of a mammalian heart can be divided into five stages based on molecular and physiological characteristics: commitment, septation, regional specification, terminal differentiation and adaptation (Melnik et al, 1995; Schott and Morrow, 1993; Chien et al, 1993). The heart originates from a lineage of mesodermal-derived progenitor cells which develop into a primordial heart tube (commitment). This process can be divided into specification and determination. Specification is the stage at which cells or tissue fragments differentiate according to their known fate when cultured in a neutral environment. Determination is the stage at which cells or tissue fragments can differentiate irreversibly to a particular developmental fate even in a novel or an ectopic environment, such as when placed in an ectopic location in the embryo. For the cardiac mesoderm, specification and determination occur at postgastrulated stages 4 to 7 and stages 7 to 8, respectively. The heart tube further develops to form distinct cardiac chambers and to gain regional-specific properties of atrial, ventricular and conducting cells (differentiation and diversification). Differentiation can be determined experimentally by studying the expression of cardiac specific genes, such as ventricular myosin heavy chain 1 ( V M H C 1 ) or cardiac troponin I, whose m R N A s and protein products are detected as early as stages 7 to 8 (Ausoni et al, 1991). These proteins become localized to myofibrillar structures at stages 9 to 10 and actual beating is observed only after stage 10, about 10 hours after initial expression of these proteins. The cardiac-specific gene regulator appears to be cardiac muscle factor 1 (CMF1) , which has E-box dependent D N A binding activity equivalent to myoblast determination factor D (myoD) in skeletal muscle and is expressed as early as stage 8. M y o D expression is seen only during stages 6-13. Diversification of cardiac progenitor cells into atrial and ventricular lineages begins while the heart is still a 3 tubular structure. Based on Western blot analysis against chamber-specific myosin heavy chain isoforms, the progenitor cells diverge to a distinct cell lineage at stages 12-15 when the heart tube begins to loop and to form distinct chambers. The molecular mechanism for this is not yet known but the position of progenitor cells, anterior vs. posterior, appears to be a key factor for determination of ventricular and atrial myocytes. Following the structural formation, the embryonic heart grows as the number of cells increases (proliferation), for about 2 weeks after birth, after which myocytes cease proliferating and grow only in size (terminal differentiation). Under some pathological and physiological conditions, differentiated adult cardiac myocytes can abnormally over-grow in size (hypertrophy, adaptation) (Chei, 1993). Even though this may be an adaptive process to compensate for the increased cardiac workload, it is regarded as an independent and significant risk factor for sudden death, myocardial infarction, congestive heart failure, and other types of cardiovascular morbidity and mortality (Levy et al, 1989). v 1.2.1. Physical and structural aspects of postnatal development of the heart. During postnatal development, the heart rapidly grows in size and weight. In rat, the weight of the newborn heart increases 35-fold by maturity and the shape becomes more spherical with age (Lee et al, 1975). The relative growth rate of heart to body weight (a) is highly proportional (a=L01) during the first month of age, but it declines in later heart growth (a=0.70). Accordingly, the ratio of heart weight to body weight decreases from about 0.5% in the newborn to about 0.2% in the adult (Rakusan, 1984). Structurally, cardiomyocytes make up 25% of the total cells and 80% of the total mass and volume of the adult heart (Zak, 1984). The blood vessels, nerves and branches of the conducting systems lie between the sub-endothelial tissues and myocardium, as well as between the myocardium and epicardium. These cells and other cells in interstitial space of ventricular tissue comprise the other 20% of the heart weight and volume. The interstitial space of ventricular tissue is filled with extracellular matrix and connective tissue cells. The connective cells consist of fibroblasts, undifferentiated mesenchymal cells, Anitschkow cells, histiocytes, macrophages and mast cells. The remaining non-cardiomyocyte mass includes collagen, vascular elements and proteoglycans. The age-related changes in the proportion of cardiomyocytes to other cells remains constant (about 80%) in canine heart but in rat the value decreases from 86% at birth to 75% in day 11 of the postnatal period (Anversa et al, 1980). Accompanying the physical changes, the structure of the cardiomyocyte is also modified during postnatal development. Cardiac muscle is composed of quasi-cylindrical cross-striated fibers connected to neighboring fibers by bifurcation. Each fiber is composed of several myocytes joined end-to-end or end-to-side via specialized junctional apparatus called intercalated discs. The dimensions of adult cardiac myocytes are relatively similar throughout most species and differences in cardiac size result from changes in cell numbers. However, in human and rabbit hearts, left ventricular myocytes have larger diameters than right ventricular myocytes, while these differences are not observed in rat hearts. The length of rat cardiomyocytes grows from 22 um to 44 um, and the diameter grows from about 5 um to about 12 um during the first 11 postnatal days (Rakusan, 1984; Zak, 1974). Average cell volume triples during this period and 5-fold by maturity (Anversa et al, 1980). 5 B y volume, adult left ventricular myocytes of rat are occupied primarily by myofibrils (50-60%), and mitochondria (30-35%) (Canale et al, 1986a). The nucleus constitutes between 1.1-2.8% of total cell volume and 5.2% is matrix. The T-tubule system and sarcoplasmic reticulum occupy 0.8-1.6% and 0.9-4.9%, respectively. At birth, myofibrils are sparse, constituting about 37% of cell volume and located at the periphery of the cell, and sarcomeres are incomplete with disoriented M , I and A bands. T-tubules are absent at birth (Carlsson et al, 1982) and are first seen at 21 days in rat ventricle (Page et al, 1974). With few regions of specialized cellular contact between myocytes at birth, the intercalated discs are few in number, short in size, and not interdigitated (Canale et al, 1986b). The sarcoplasmic reticulum (SR) is seen at birth, but volume increases postnatally. The surface area ratio of SR to myofibrils increases during the first 21 days but remains constant afterward, indicating proportional growth of these components (Riva and Hearse, 1991). Mitochondria are small, irregular in shape, relatively few in number, sparse in cristae, and randomly oriented at birth. A t maturity, they become enlarged, uniform, and aligned in rows between myofibrils (Anversa et al, 1973). At the same time, the myocardial glycogen content declines up to 86% in the 17 t h postnatal day (Shelley, 1961). The percentage of bi-nucleated myocytes increases from 3% at birth to 50% at 11 days of age (Anversa et al, 1980) and the total number of nuclei in the left ventricle of the rat heart increases from 7.5 x 10 6 in 1 day to 13 x 10 6, 22 x 10 6, 30 x 10 6, and 40 x 10 6, respectively, in 5, 11, 21, and 60 days after birth (Anversa et al, 1986). The total number of the rat left ventricular myocytes, however, doubles from 7.5 million to 14 million in the first 10 days of postnatal period and to 30 million at the age of 60 days, which then stays in constant afterward (Rakusan, 1984; Zak 1984b; Anversa et al, 1980), indicating that some of the ventricular myocytes become multi-6 nucleated during postnatal development. The coronary vascular bed in the rat heart continues to develop until around the 12 postnatal day (Rakusan, 1984). The postnatal development of coronary vessels refers to the formation of the coronary artery branches in the atria and the formation of intercoronary anastomoses. After the 12 t h postnatal day, no new arterial stems or anastomoses are formed and the vascular system already established only adapts itself to the growing heart. The autonomic nervous system of the rat heart also develops substantially after birth in morphology and physiology. Regarding sympathetic innervation, the inotropic response to adrenergic innervation only develops after 2 to 3 weeks of age, even though functioning p1-receptors and adrenergic fibers are detected in newborn heart (DeChamplain et al, 1970). Similarly, the concentration of endogenous norepinephrine is very low at birth, but it increases rapidly during the first week. B y comparison, cholinergic innervation develops earlier than sympathetic innervation. Cholinesterase activity is detectable in rat heart before birth (Finlay and Anderson, 1974) and is functionally mature in the short period after birth, at about the 10 t h day of age (Vlk and Vincenzi, 1977; Taylor, 1977). The amount of acetylcholine also increases after birth, concomitant with an increase of choline acetyltransferase activity (Rakusan, 1984). 1.2.2. Molecular studies on the mechanism of cardiac myocyte terminal differentiation. The regulatory mechanisms of cardiac development and growth are still scarcely understood. One of the main obstacles in investigating cardiac growth is the inability of adult cardiac myocytes to proliferate while maintaining a differentiated phenotype in culture. However, neonatal myocytes can 7 undergo one or two cycles of mitosis before they become terminally differentiated. Therefore, most studies are limited to the culture of neonatal cardiac myocytes, which are distinct from the adult cardiac myocytes in many ways. Current understanding of the genetic programing of cardiac myocytes is largely based on studies of skeletal myoblasts, in which the morphology and biochemistry are assumed to be similar (Olson, 1992). Among the various transcription factors, M y o D and related proteins, including myogenin, myogenin factor 5 (Myf-5), muscle-specific enhancer binding nuclear factor 1 (MEF-1) and muscle regulatory factor 4 (MRF-4), appear to play important roles in generating muscle-specific phenotypes (Weintraub et al, 1991; Edmonson and Olson, 1993; Lassar et al, 1994; Ross et al, 1996). These proteins commonly have a basic helix-loop-helix (bHLH) motif, followed by a basic region, which is involved in D N A binding (Davis, 1990). These muscle-specific proteins form heterodimers with other ubiquitous b H L H proteins and activate the muscle gene program (Murre et al, 1989; Navankasattusas et al, 1994). E12 and E47, which are splice variants of the E2 gene, are the dimerization partners of the MyoD-like b H L H proteins (Murre et al, 1989b; Blackwell and Weintraub, 1990). The heterodimer has a high affinity to C A N N T G (N refers to an ambiguous sequence) D N A sequences (E-box sites), which are located in the 5'-flanking and/or intron regions of several muscle-specific genes, such as myosin light chain 2 (MLC-2) (Navankasattusas et al, 1994), cardiac a-actin (Moss et al, 1994), and a-myosin heavy chain (a-M H C ) (Molkentin, et al, 1993). Another ubiquitous b H L H protein, Id (inhibitor of differentiation), which lacks the basic D N A binding regions, can form complexes with MyoD-like proteins and play inhibitory role in gene transcription. Even though E l 2 , E47 and Id have been detected in cardiomyocytes, MyoD-like proteins or cardiac-specific b H L H proteins have not been detected (Olson, 1992). 8 Transcriptional enhancer factor 1 (TEF-1) is another class of factor that binds to the M - C A T sequence ( C A T T C C T , muscle-CAT heptamer) for regulation of the cardiac troponin T gene (Mar and Ordahl, 1990). In the mice, ablation of the TEF-1 gene has been shown to result in fetal death due to a defect in cardiac maturation (Chen et al, 1994). The hybrid of E-box and M - C A T , known as EM-box, has been shown to induce a - M H C gene expression in response to c A M P K (Gupta et al, 1996a). Recently, it has been shown that TEF-1 interacts with a b H L H leucine zipper protein, Max, and positively regulates the expression of a - M H C gene through the EM-box (Gupta et al, 1997). A n A and T-rich D N A sequence (CC(A+T)6GG, cis regulatory element) has been found in the promoter/enhancer regions of various muscle genes, such as creatine kinase ( M C K ) , cardiac troponin T (cTNT), myosin light chain-2 (MLC-2) , and phosphoglycerate mutase (Zhu et al, 1991; Gossett et al, 1989). These findings indicate the presence of E-box-independent pathways in cardiac gene regulation. Studies of the promoter region of the M L C - 2 (myosin light chain isoform) gene have revealed that the first 250 base pairs of the promoter confer cardiac-specific expression and contain three highly conserved regions: HF-1, HF-2 and HF-3 (Zhu et al, 1991). Among these conserved regions, the HF-1 site (28 base pairs) is involved in the cardiac specific gene expression of M L C - 2 , in response to a-adrenergic receptor activation (Zhu et al, 1991). Two transacting factors, HF-1 a and H F - i p , were found to interact with the HF-1 site (Navankasattusas, 1992). H F - l a is ubiquitous and binds to the core region of the HF-1 site, whereas HF-1 (3 binds to the A and T rich region and is expressed only in terminally differentiated skeletal muscle, cardiac muscle and brain. H F - i p contains three zinc finger motifs that may interact with D N A . Another A and T rich region^ known as myocyte enhancer factor-2 binding site (MEF-2 site), is found in muscle creatine kinase ( M C K ) enhancer (Gossett et al, 1989; Navankasattusas, 1992). H F - i p is different from M E F - 2 site binding factors 9 that are members of the family of serum response factor genes. To date, little is known about the intrinsic and extrinsic factors that are responsible for the growth and terminal differentiation of cardiac myocytes. Skeletal muscle myoblasts remain in the undifferentiated proliferative state when high levels of serum are maintained in the culture media. This effect appears to be mediated by several factors, including the enhanced expression of Id, which prevents dimerization of M y o D and E12 (Brennan, 1991; Jen et al, 1992), expression of proto-oncogenes, such as Fos, Jun and Myc, which inhibit M y o D dimerization (Bengal, 1992), inhibition of myogenic b H L H factors by inhibitory phosphorylation (Hardy et al, 1993), and direct or indirect inhibition by cyclin D-dependent kinases (Skapek et al, 1995). During the development of the rat heart, the decrease of c-myc expression is synchronous with the terminal differentiation of the cardiomyocyte and an overexpression of c-myc in transgenic mice results in cardiac enlargement due to increased myocyte hyperplasia during the fetal period (Machida et al, 1997). Among the genetic markers for differentiated cardiomyocytes, myosin heavy chain family has been one of the best characterized. In rat heart ventricle, two myosin heavy chain genes (a and (3) are expressed. The proteins encoded by these genes complex to form three isoforms of myosin (referred to as V I , V 2 and V3). The V I isoform comprises a - a homodimers, whereas V3 and V 2 are (3—(3 homodimers and a-p 1 heterodimers, respectively. During fetal life, the V 3 isoform is the most abundant myosin in most species (Lompre et al, 1984). The V I isoform appears after birth and the level of expression gradually declines with age (Swynghedauw, 1986). Thyroid hormone and catecholamines both contribute to the up-regulation of V I and down-regulation of V3 isoforms (Sweadner, 1992; Gupta et al, 1996a; Gupta et al, 1996b). The a subunit of Na/K-ATPase also 10 undergoes changes in isoform expression in the neonatal heart (from oc3 to al), which is also regulated by thyroid and glucocorticoid hormones (Sweadner, 1992; Lompre, 1984). Insulin-like growth factor 1 (IGF-1), which acts in autocrine or endocrine fashion, influences the growth of myocytes during maturation. During the first few days of postnatal life, the expression of IGF-1 and IGF-1 receptors (IGF-1R) in myocytes sharply decreases. This parallels the decrease in D N A replication and cell proliferation (Anversa et al, 1996a). In addition to the temporal regulation of IFG-1, several types of indirect evidence have supported the role of IGF-1 in cardiac proliferation. One day old cardiac myocytes proliferate and undergo cellular hypertrophy in the presence of 10% fetal calf serum (FCS). IGF-1 (100 ng/ml) without FCS can also support proliferation without causing cellular hypertrophy (Kajstura et al, 1994). Furthermore, treatment with antisense oligodeoxynucleotides to IGF-1R m R N A inhibited the FCS induced D N A replication and mitotic division of myocytes (Anversa et al, 1996a). These observations indicate that activation of IGF-1 R in myocytes is required for D N A synthesis and cellular hyperplasia. 11 1.3. Three paradigms for protein kinase signal transduction mechanisms and their roles in postnatal development of the heart. Many neurohormones and growth factors target the heart, modulating growth and function. Binding of these ligands to their receptors induces conformational changes in the receptor, which can induce activation of various proteins by protein-protein interactions or reversible phosphorylation. Protein kinases are enzymes which catalyze transfer of a y-phosphate of A T P to a substrate protein, which often changes the structural conformation and modulates the activity of the substrate. When the target is another protein kinase, this phosphorylation often can regulate its activity. These sequential events of phosphorylation are one of key mechanisms by which cells disseminate and amplify extracellular stimuli into intracellular functional targets in organisms. The mechanisms of protein kinase activation can be divided into three paradigms (Fig. 2). The first paradigm which applies to protein kinases such as calmodulin-dependent protein kinases (CaMPK), cyclic A M P -dependent protein kinase (cAPK), and cyclic GMP-dependent protein kinase (cGPK), which are directly activated by various second messengers such as C a 2 + , cyclic A M P or cyclic G M P , respectively (Fig. 2A). These protein kinases can either directly phosphorylate and regulate functional targets or modulate other signalling cascades. They are utilized for signals that need to be rapid and of short duration. For example, phosphorylation of phospholamban by C a M P K 2 in cardiomyocytes is a key-signalling cascade modulating contractility in a short period. The second paradigm involves the sequential activation of a protein kinase by seryl, threonyl or tyrosyl phosphorylation by another protein kinase with a cascade, as occurs for the mitogen-activated protein (MAP) kinase and S6 kinase pathways (Fig. 2B). These signalling cascades not 12 Figure 2. Three paradigms for signal transduction mechanism in prokaryotes and eukaryotes. The diagram A indicates that the protein kinase is activated by directly binding to second messengers such as Ca2+, c A M P and c G M P . The diagram B shows the signalling cascade mediated by multi-tiers of sequentially activating protein kinases. The diagram C illustrates the signalling pathways mediated by the stepwise transfer of a phosphate group between proteins. 13 only increase the sensitivity to extracellular stimuli by amplifying the intracellular signals but also can convert a graded stimulus into a switch-like response (Ferrell, 1996). For example, in the Ras-Raf-Mek-Erk M A P kinase cascade, there are about 20,000 molecules of Ras and about 1,000,000 molecules of E r k l and Erk2 per cell. It has been estimated that the 2,000 to 10,000 molecules of active Ras can activate almost all of the E r k l and Erk2. This corresponds to about 100- to 500-fold amplification (Ferrell, 1996). The last paradigm is a cascade with multiple layers of linear phosphorylations on histidyl and aspartyl residues from receptors to functional effectors. The only known example is the two-component regulatory systems in prokaryotes and low eukaryotes (Fig. 2C). This type of signalling module has no amplification in response and may be advantageous for signals that are to be tightly regulated. During the early postnatal period, cardiomyocytes undergo significant changes in physical and biochemical characteristics, including proliferation/differentiation, metabolism, contractile function, and response to hypoxia and ischemia (Rakusan, 1984; Zak, 1984a; Michalak, 1987; Rumyantsev, 1977). In addition to the molecular changes resulting in the progressive expression of cardiac-specific genes, various signal transducing proteins are regulated by the expression and post-translational modifications during development of the heart (Chien et al, 1991; Chien et al, 1993; Cummins, 1993; Schott and Morrow, 1993; Van-Bilsen and Chien, 1993). For example, the increased sensitivity of (3-adrenergic signals in the adult heart is linked to the down-regulation of a specific isoform of trimeric G proteins (Gi, inhibitory). The level of expression of G i declines by as much as 6-fold by 30 days of age, without a change in the level of Gs (stimulatory) expression (Hansen, 1995; Battel et al, 15 1996). In addition, expression of type V isoform of adenylyl cyclase (AC) increases during postnatal development and is involved in the higher basal A C activity in adult heart (Espinasse et al, 1995; Tobise et al, 1994). The increase in cardiac troponin I expression is also implicated in the higher efficiency of ^-adrenergic stimulated relaxation of adult ventricles (Rakusan, 1984). 1.3.1. The second messenger- and cyclin-dependent protein kinases (CDKs) in proliferation and differentiation of cardiomyocytes. In rat heart, terminal differentiation occurs about two weeks after birth, myocytes being arrested at Go or G i phase of the cell cycle (Capasso et al. 1992; Zak, 1974). The rate of D N A synthesis and the D N A polymerase activity decline rapidly in the same time period (Claycomb, 1975). Recently, the bromodeoxyuridine (BrdU) incorporation assay indicated that about 17% of the left ventricular myocytes of rats were labeled at birth, and this value decreased to 13% at 1 day after birth and 0.2% at 2 months of age (Cheng et al, 1995). As discussed in a previous section, the cardiac mass increases as much as 35-fold from birth to adulthood, but the number of left ventricular myocytes only increases 2- to 3-fold, from about 7 x 106 cells at birth to 14 x 10 6 and 30 x 10 6 at the age of 11 and 60 days, respectively (Rakusan, 1984; Zak, 1984b; Anversa et al, 1986). This number remains constant in the later stages of development. While it is not yet clear how many times a cardiac myocyte undergoes mitosis during early postnatal development, it has been suggested that actual mitosis may take place more than twice in view of the number of cells that are also subject to apoptosis during postnatal development (Misao et al. 1996; Kajstura et al. 1996). Isolated 5-10 day old neonatal cardiac myocytes undergo one or two cycles of cell division before becoming differentiated in vitro. Adult cardiomyocytes grow only in size (hypertrophy) and occasionally become bi- and/or poly-nucleated. Under some pathological conditions, cardiac myocytes further 16 grow in size and the number of bi- or poly-nucleated cells increases. The mechanism(s) of terminal differentiation is still unknown, but it appears to result from the biochemical inhibition of mitosis and D N A synthesis rather than the permanent physiological loss in their ability to proliferate. Re-initiation of D N A synthesis and mitotic division of adult cardiac myocytes under certain conditions, such as ischemia (Anversa, et al, 1996b), pressure overload (Capasso et al. 1993), anemia (Olivetti et al. 1992), phorbol ester treatment (Claycomb and Moses, 1988), and in senescent heart in certain strains of rats (Anversa et al. 1991) substantiate this postulation. A temporal cue for the terminal differentiation might be provided by development of adrenergic nerves in the early postnatal period causes the inhibition of D N A synthesis (Claycomb, 1975; Claycomb, 1976). The activity of cyclic-AMP-dependent protein kinase ( cAMPK) peaks in the 7 day-old neonate, and then progressively declines with age (Haddox et al. 1979). In contrast, the intracellular concentration of cyclic-GMP and cyclic-GMP-dependent protein kinase activity continuously decline during late fetal and early postnatal development (Claycomb, 1975; Kuo, 1975b). Expression of Goq, G a i i , phospholipase C (PLC), and protein kinase C (PKC), which are closely involved in cell growth and proliferation, are also down-regulated during development (Puceat et al. 1994; Rybin and Steinberg, 1994; Harada, 1995). Studies with skeletal myocytes have provided clues for the underlying mechanism of the teirninal differentiation by using a group of D N A tumour viruses such as SV40, polyoma, and adenovirus, which can inhibit and reverse the terminal differentiation (Endo and Nadal-Ginard, 1989). The cardiac myocytes of transgenic mice expressing the SV40 large T-antigen oncoprotein were hyperplasic while retaining the characteristics of differentiated cardiomyocytes (Katz et al. 1992; Field, 1988). The oncoprotein SV40 T-antigen was found to complex with a group of tumour suppressor proteins, 17 such as retinoblastoma (Rb), p53, pl07 and pl30 (Daud et al. 1993), the activities of which are modified by various cyclin-dependent kinases (CDK) (Sherr, 1994; Weinberg, 1995). It has been well established that G l cyclins with their catalytic partners, the C D K s , can phosphorylate and inactivate the group of tumour suppressor proteins (Weinberg, 1995; Skapek et al, 1995; Skapek et al, 1996). These studies have demonstrated that the expression of cyclins A , D l , D2 and E inhibits muscle gene expression in fibroblasts expressing M y o D and in C2C12 myoblasts, and this inhibition can be reversed by expression of a nonhyperphosphorylatable form of Rb (except by cyclin D l , which may indicate the presence of additional Rb-independent inhibitory mechanisms of cyclin D l ; Skapek et al, 1996). In fact, the expression of Rb was increased and the protein became hypophosphorylated upon muscle differentiation (Endo and Goto, 1992; Martelli et al, 1994). Furthermore, MyoD-mediated transactivation of muscle gene in SASO-2 osteosarcoma cells, which lack functional Rb, requires coexpression of wild-type Rb or pl07, indicating that Rb and related proteins regulate M y o D function (Gu et al, 1993; Schneider et al, 1994). During the skeletal muscle differentiation, several C D K inhibitors are induced, including p ^ 1 1 ^ 4 , a specific inhibitor of cyclin-D/CDK4 and C D K 6 kinases, and p 2 1 C I P 1 / W A F 1 , a broader C D K inhibitor (Parker et al, 1995b). At the same time, ectopic expression of these inhibitors facilitates muscle differentiation (Serrano et al, 1993; Xiong et al, 1993) and stumulates M y o D function in serum-stimulated myoblasts (Skapek et al, 1995). However, in Rb-deficient myocytes, ectopic expression of C D K inhibitors neither augments M y o D function nor prevents S phase entry (Novitch et al, 1996), indicating that a primary role of C D K inhibitor in skeletal muscle is to maintain the hypophosphorylated, activated state of Rb. In addition, differentiated myocytes lacking Rb accumulate in the S and G2 phases of the cell cycle and fail to proceed to mitosis. Further, these cells undergo mitotic catastrophe once C D K s are activated by removing phosphate from inhibitory phosphorylation sites (Novitch et al, 1996). These results 18 indicate that active Rb plays an important role in preventing differentiated myocytes from entering the S and G2 phases, whereas the inhibition of mitosis is independent of Rb activity. 1.3.2. Mitogen-activated protein kinase (MAPK) signalling pathways in cell proliferation/differentiation and ischemia of cardiomyocytes. Mitogen-activated protein (MAP) kinases are a family of proline-directed Ser/Thr kinases involved in various signalling pathways that mediate cell growth, differentiation, transformation, cellular stress responses and apoptosis (Su and Karin, 1996; Davis, 1995; Errede et al. 1995; Morrison, 1995; Waskiewicz and Cooper, 1995; Pelech and Sanghera, 1992; Sugden and Bogoyevitch, 1995a; Robinson and Cobb, 1997). Extracellular signal-regulated kinases (Erk) 1 and 2 are M A P kinases that are involved in mitogenic signals by phosphorylating various proteins. Erk substrates include 90-kDa ribosomal S6 protein kinases (Rsk) and several nuclear proteins such as c-TCF Myc, c-Jun, NF-IL6 (a transcription factor), p62 (one of the Elk-1 complex proteins that bind to serum response element of D N A ) and ATF-2 (a transcription factor that binds to c A M P response element; CRE) . For maximum activation of E r k l and Erk2, phosphorylation is required at both Thr and Tyr residues, located in a ' T E Y " motif before the kinase catalytic subdomain VIII region, by a dual specificity protein kinase known as Mek ( M A P kinase/Erk kinase kinase). Both receptor tyrosine kinases (RTK) and seven transmembrane domain receptors (STMR) can activate Mek via the monomeric 21-kDa G protein, Ras (Bokoch, 1996; Post and Brown, 1996). Upon ligand binding to R T K , the dimerized R T K cross-phosphorylates and attracts growth factor receptor binding protein-2 (Grb2) via its Src homology 2 (SH2) domain to the tyrosine-phosphorylated sites. Grb2 is an adapter protein that is also bound to the guanine nucleotide exchange factor SOS through its SH3 domains. SOS promotes binding of G T P to Ras, resulting in its activation. GTP-Ras recruits Rafl to the 19 plasma membrane where Rati is activated by phosphorylation by other protein kinases, including P K C and Src. The activated Rafl phosphorylates and activates M e k l and Mek2, which can sequentially activate E r k l and Erk2, and R s k l and Rsk2. While the activation of Ras by R T K has been delineated in detail, the signalling steps connecting S T M R to Ras are less clear. Figure 3 depicts the putative signalling pathways from the S T M R to M A P kinases. The STMRs interact with specific classes of heterotrimeric G proteins and activate mitogenic signalling cascades by multiple mechanisms (Hawes et al, 1995). Among them activation of Ras has been shown to be one major pathway for the stimulation of mitogenesis by several STMRs, including the muscarinic m l and m2, a2-adrenergic, angiotensin II, endothelin-1, thrombin and lysophosphatidic acid receptors (Winitz et al, 1993; Howe and Marshall, 1993; Cook et al, 1993; Alblas et al, 1995). Generally, the mitogenic response to these receptors appears to be mediated by either G i or Gq (Gutkind, 1998). Evidence provided by various laboratories has also established that the activation of M A P K cascade by G i is mediated by G protein py subunits (Koch et al, 1994; Crespo et al, 1994; Ito et al, 1995), probably, by Gpy-mediated tyrosine phosphorylation of She (van Biesen, et al, 1995). She, existing as three isoforms with apparent molecular masses of 46-, 52- and 66-kDa, is an adaptor protein containing a single SH2 domain and a proline-rich SH3-binding motif. The tyrosine phosphorylation of She mediates binding to other SH2-containing proteins, such as Grb2 (Pawson and Schlessinger, 1993), which then activates Ras and Erks by the mechanism discussed earlier. The phosphorylation of She is proposed to be mediated by activation of Src and Src related kinase, Lyn, and possibly others, while the mechanism of the Src activation by Gpy is unknown (Wan et al, 1996; Schieffer et al, 1997). Bokoch (1996) speculated that Gpy communicates with Src via a pleckstrin homology (PH) domain-containing protein, based on 20 Figure 3. Putative signalling pathways in the activation of MAP kinases by seven transmembrane domain receptors. Solid arrows indicate established protein-protein or protein phosphorylation interactions. Dotted arrows indicate that the presence of relationship in signalling pathway is observed but the mechanism of interaction is still to be delineated. For abbreviations, see List of abbreviations. See pages 20-27 for details of the relationships between these signalling proteins. 21 STMRs STMRs ^ ^ ^ < - > ^ ^ ^ ^ ^ j ^ l ^ l 2 2 observations that Gpy binds a P H domain of other signalling molecules (Inglese et al, 1995) and a wide variety of guanine nucleotide exchange factors (GEF) for small G proteins (McCollam et al, 1995). While Ras was shown to bind directly to the Gpy subunit in vitro (Xu et al, 1996), the direct in vivo evidence is still lacking. In addition, wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, can diminish Erk activation by STMRs (Hawes et al, 1996) and a novel PI3K isoform (PDRy), which is activated by Gpy, plays a critical role in the activation of Erks by Gi-coupled STMRs (Lopez-Ilasaca et al, 1997). Angiotensin II (ANG-II) and endothelin-1 (ET-1) receptors have also been shown to activate M A P kinases via P K C , but are independent of the activation of Ras (Arai and Escobedo, 1996; Zou et al, 1996; Takahashi et al, 1997). Several studies have documented that the activation of M A P kinase by GPy subunits requires neither P L C - p nor P K C activation, but is blocked by inactive Ras (Koch et al, 1994; Crespo et al, 1994). Interplay between P K C and Ras has been shown in various cells. In T cells, activation of P K C is accompanied by significantly increased Ras activity (Downward, et al, 1990). It has been shown that expression of a constitutively active Ras gene also results in an elevation of diacylglycerol, which would then be expected to chronically activate P K C (Wolfman et al, 1987). Studies with over-expressed GTPase activating protein (GAP) and immunoprecipitation indicate that the activation of Ras by P K C occurs through phosphorylation of G A P or GAP-associated proteins (Nori et al, 1992; Blackshear et al, 1992). In addition to Ras, annexin-like protein, 14-3-3, appears to interact with Rafl (Freed et al, 1994; Irie et al, 1994; Fu et ah, 1994) but its role is still unclear. The association of Rafl and these binding proteins may facilitate its phosphorylation at multiple sites by other protein kinases, including Tyr-340 (a putative activating phosphorylation residue) resulting in activation of M e k l and Mek2 (Morrison et al, 1993). Protein kinase C was shown to directly phosphorylate and 23 activate Rafi by phosphorylating another site, Ser-499 (Siegel, et al, 1990). Mutation of this site to alanine blocked P K C mediated activation without affecting activation by a combination of Ras and a Src-related kinase, Lck (Kolch et al, 1993). In addition to Raf i , two other isoforms, Raf A and RafB with distinct cellular distributions and characteristics have been identified and characterised (Daum et al, 1994; Boldyreff and Karin, 1997; Hagemann et al, 1997). Besides Raf, other Ser/Thr protein kinases have been shown to phosphorylate and activate Mek. These include the 78-197-kDa Mek kinases (Mekks, mammalian homologues of yeast STE11; Lange-Carter et al, 1993; Blank et al, 1996), the 52-58-kDa oncogene-encoded Ser protein kinase Tpl2 (Salmeron et al, 1996), the 34-kDa Mos (Nebreda et al, 1993; Pham et al, 1995) and K S R (kinase suppressor of Ras; Therrien et al, 1996). The physiological roles and mode of activation of these kinases are less apparent. The truncated forms of Mekk and Tpl2 are known to be constitutively active (Xu et al, 1995; Kovatch et al, 1997). In fact, the N-terminal truncation of Mekk 1 by caspase-3 is required for its activation (Cardone et al, 1997). Mos was proposed to be activated by P K C (Kroll et al, 1991) and inactivated by binding to casein kinase 2 (CK2) (3 subunit (Chen, et al, 1997). However, the exact mode of activation of these kinases in the heart still remains to be delineated. In cardiac myocytes, most of these mitogenic signal transduction pathways seem to be conserved. The activation of E r k l and Erk2 by various hypertrophic stimuli via Mek has been shown in both neonatal and adult ventricular cardiac myocytes. Introduction of constitutively active Ras or estradiol-regulated Rafi leads to activation of E r k l and Erk2 and induces several hypertrophic markers (Thorburn et al, 1993; Thorburn et al, 1994a). Similarly, expression of dominant inhibitory Ras or 24 Rafl mutants blocks both the activation of Erk l /2 and expression of genetic markers induced by phenylephrine. However, the hypertrophic-signalling pathway seems to be more complex than Ras —> Raf —» Mek —> M A P kinase. Transfection of dominant inhibitory E r k l or active Rafl into neonatal ventricular cardiac myocytes neither inhibits nor enhances the induction of actin incorporation into sarcomeric units, which is one of the hypertrophic markers (Thorbum et al, 1994b). However, active Ras is able to induce these changes (Thorbum et al, 1993). These observations implicate the involvement of another G protein family, such as Rho, which appears to mediate growth factor effects upon the cytoskeletal actin organization in cardiac hypertrophy. Studies employing phorbol esters and constitutively active P K C have revealed involvement of P K C in the activation of M A P kinases and induction of hypertrophic markers (Shubeit et al, 1992; Kariya et al, 1994). However, activation of M A P kinases by endothelin-1 (ET-1), angiotensin II (ANG-II) and epidermal growth factor (EGF) is only partially inhibited by phorbol ester-induced P K C down-regulation. Furthermore, the activation by basic fibroblast growth factor (bFGF) and tumour growth factor (3 (TGFp) is not affected at all by P K C down-regulation (Cummins, 1993). The involvement of C a 2 + in the mitogenic signals has also been indicated in various cell types (Pribnow et al, 1992). In cardiac myocytes, the calcium ionophore A23187 activates E r k l and Erk2, and the calcium chelater B A P T A completely abolishes ANG-I I induced activation of these kinases (Sadoshima et al, 1995). In addition to Erk 1 and Erk2, several other MAPK-related proteins have been identified. One subfamily of M A P K is the stress activated protein kinases (SAPKs), comprising at least three isoforms: S A P K a , (3 and y (also known as c-Jun N-terminal kinase (JNK) 2, 3 and 1, respectively). Various stress-inducing stimuli such as heat shock, U V radiation, tumor necrosis factor-a and the protein synthesis inhibitors, cycloheximide and anisomycin, activate these protein kinases (references within Pelech and Charest, 1995). SAPKs in turn stimulate the activity of several transcription factors 25 including c-Jun, ATF2 , Elk and Sap-1 (Cavigelli et al, 1995; Gupta et al, 1995; Whitmarsh et al, 1995). Another M A P kinase subfamily is related to the 38-kDa yeast Hog (high osmolality glycerol)l kinase, and is also referred to as reactivating kinase (RK) or kinase p40 (Freshney et al, 1994). This protein kinase group includes the isoforms p38a, p38p\ p38y and p388 (references within Enslen et al, 1998), and they are activated by various stimuli such as lipopolysaccharide, hyperosmolar medium, taxol, arsenite, interleukin 1 and T N F - a (references within Pelech and Charest, 1995). In various cells, the activation of p38 has been implicated in gene regulation, morphological alterations, and cell survival in response to stresses, which is in part mediated by phosphorylating and activating transcription factors C R E B , A T F 1 , ATF2 , C H O P and M E F - 2 C (Tan et al, 1996; L in et al, 1997; McLaughlin et al, 1996; Wang and Ron, 1996). It can also activate various protein kinases by direct phosphorylation, including M A P kinase-activated protein kinase ( M A P K A P K ) - 2 , -3 (McLaughlin et al, 1996) and -5 (Ni et al., 1998), M A P kinase signal-integrating kinase (MNK)-1 and -2 (Waskiewicz et al, 1997), and p38-regulated/activated protein kinase ( P R A K ; New et al, 1998). The activation of M A P K A P K - 2 , -3 and P R A K in turn can phosphorylate the small heat-shock proteins, hsp25 and hsp27, which are involved in the stablization of actin microfilament networks upon stress (Landry and Hout, 1995; Guay et al, 1997). Activated M N K 1 can phosphorylate translation initiation factor eIF-4E, suggesting a role in translation (Waskiewicz et al, 1997). Upstream regulators of these M A P kinases appear distinct from those of E r k l and Erk2. Various studies indicate that S A P K s are activated by 43-kDa Sekl (SAPK/Erk kinase, also called Mkk4 or J N K K 1 ; Salmeron et al, 1996), J N K K 2 (Lu et al, 1998) and Mkk7, which may in turn be regulated by M e k k l (Lange-Carter et al, 1993) or mixed lineage kinase 3 (Mlk3) (Ranna et al, 1996). Activation of Hog may be mediated by Sek l , Mkk3 and Mek6 (Moriguchi et al, 1996) which may lie downstream from p21-activated kinases (Pak) (Bagrodia et al, 1995; Zhang et al, 1995; 26 Jakobi et al, 1996; Teramoto et al, 1996). Pak activity is directly regulated by the binding of either of two related small G proteins, Rac and Cdc42 (Manser et al, 1994, Zhang et al, 1995). To date the mechanism for the activation of these stress-regulated signalling pathways, I N K and p38, by S T M R s have not yet been fully delineated. Figure 3 illustrates the putative signalling pathways involved in the regulation of p38 by S T M R . The chemoattractant receptors of leukocytes can activate Pak through the pertussis toxin-sensitive Gi protein (Knaus et al, 1995). Rac and Cdc42 can be activated by G(3y subunits via PH-domain containing Rho small G protein specific GEFs , such as Lfc and Lsc, analogous to the mechanism of Ras activation by G[3y (Glaven et al, 1996; Michiels et al, 1997). However, Lfc binds to Rac regardless of the guanine nucleotide binding state (either G T P - or GDP-bound Rac), which may indicate other roles of Lfc in the regulation of Rac (Glaven et al, 1996). In addition, PI3Ky is also stimulated by Gpy and is involved in the activation of J N K via Rac and Pak (Lopez-Ilasaca et al, 1998). Besides activation of Pak by Rac and Cdc42, M e k k l and 4 are specifically associated with Rac and Cdc42, and kinase-inactive mutants blocked Rac/Cdc42 stimulation of J N K activity (Fanger et al, 1997b; Gerwins et al, 1997). As mentioned earlier, M e k k l is a substrate for proteolytic cleavage by caspase and is capable of inducing apoptosis (Cardone et al, 1997). The inhibitory mutants of M E K K 1 and 4 did not affect Pak activation of J N K , indicating that Pak and Mekk independently activate J N K . During postnatal development of the heart, a decrease in resistance to ischemia is another significant change. The neonatal heart possesses a greater resistance to ischemia than the adult heart, showing greater post-ischemic recovery of contractile functions and less cellular damage 27 (Pridjian et al, 1987; Riva and Hearse, 1991; Carr et al., 1992). Several explanations have been advanced to explain for this phenomenon, including an increase in the activity of xanthine oxidoreductase in adult heart, which can generate toxic reactive oxygen intermediates during ischemia reperfusion, differences in calcium handling between neonate and adult, reduced depletion of A T P in the neonate during ischemia, and more favorable hemodynamic response during ischemia (references within Riva and Hearse, 1991; Matherne et al, 1997). 1.3.3. Activation of MAP kinases by cardiac ischemia/reperfusion and its role in cardiac preconditioning. Both the mitogenic and stress-related M A P kinases have been shown to be activated by ischemia and post-ischemia reperfusion of the heart (Bogoyevitch, 1996b; Knight 1996). Even though the mechanism of these activations still remains to be investigated, they may result in the re-initiation of D N A synthesis and mitosis of the differentiated cardio myocyte (Anversa et al., 1996b). Many genes, including immediate early genes such as c-fos, c-jun, and Egr-1 and hypertrophic markers such as atrial natriuretic factors and transforming growth factor-p1, are rapidly up-regulated during ischemia and reperfusion (Chein et al, 1993). Besides the damaging effects, such as cellular necrosis and apoptosis caused by a prolonged ischemia and reperfusion, a brief exposure of the heart to ischemia, a phenomenon known as preconditioning (PC), protects the myocardium against infarction during subsequent prolonged ischemia (Cohen et al., 1996a). Preconditioning, from a classical point of view, is induced by a critical reduction in myocardial blood flow, and the end-point is the reduction of infarct size (Murry et al, 1986). 28 However, in a broader sense, it can also be induced by a hypoxic perfusion or a reduction of coronary flow in buffer perfused heart preparations (Cohen et al, 1995), by a combination of hypoxia, substrate free perfusion, and pacing stress in isolated cardiac muscle preparation (Speechly Dick et al., 1995), or by hypoxia and lack of glucose substrate in isolated cardiomyocytes (Amstrong et al, 1994). Better recovery of contractile function (Cave, 1995) or less incidence and severity of ischemia- and reperfusion-induced arrhythmia (Hagar et al, 1991) have also been used as end-points of ischemic preconditioning. The window of cardiac protection by ischemic preconditioning appears to be bimodal. The initial phase, known as classic preconditioning, lasts about one to three hours, whereas the delayed preconditioning, also referred to "second window of protection", exists between 12 and 72 hours, depending on species and model (Marber et al, 1993). Several potential triggers of classic preconditioning have been identified, including adenosine, acetylcholine, catecholamine, angiotensin II, bradykinin, endothelin and opioids (Yellon, 1998). Intracellular signalling cascades of many of these triggers, such as noradrenaline, bradykinin and adenosine, are mediated by pertussis toxin sensitive G proteins (Thornton, et al, 1993). In both rabbit and rat hearts, P K C inhibitors block ischemic preconditioning and P K C activators mimic the effects of the preconditioning (Liu et al, 1994; Ytrehus et al, 1994; Speechly Dick et al, 1994). However, the role of P K C in the preconditioning depends on species, since P K C inhibitors fails to block the preconditioning in either dog or pig hearts (Przyklenk et al, 1994; Vahlhaus et al, 1996). A tyrosine kinase inhibitor, genistein, and A T P sensitive K + channel (K+ATP) blocker, glibenclamide and sodium 5-hydroxydecanoate, are also able to block the preconditioning (Baines et al, 1996; Amstrong et al, 1995; Auchampach et al, 1992). The identity of protein 29 tyrosine kinases subjected to genistein inhibition and their downstream signalling pathways is still to be investigated. Activation of K+ATP may reduce action potential duration, resulting in reduction of cellular C a 2 + overload during ischema. P K C activates K+ATP at near physiological intracellular A T P concentration (Light et al, 1996) and the preconditioning effects induced by the P K C activator, dioctanoylglycerol, is inhibited by glibenclamide (Speechly Dick et al, 1995). However, at a low dose, bimakalim (a K+ATP activator) exerts its cardioprotective effects without reducing action potential duration (Yao and Gross, 1994). Therefore, the role of K+ATP as an end-effector is still to be clarified. The activation both of P K C and protein tyrosine kinases may well be involved in the activation of Erk M A P kinases. Besides Erks, Mapkapk2 which is a downstream kinase for p38 M A P kinase has been suggested to play a key role in stablization of the cytoskeleton during cellular stresses (Landry and Huot, 1995; Guay et al, 1997). However, the up-stream regulator for the p38 M A P kinase remains to be identified. Another key player as an end-effector for the preconditioning may includes a critical step in energy depletion, which is coupled with proton production during ischemia and reperfusion. Details regarding metabolic considerations in preconditioning wil l be discussed in Section 1.4.3. Less information is available regarding the mode of the delayed preconditioning. Adenosine and nitric oxide (NO) appear to be important in triggering the delayed anti-infarct effects. For examples, treatment with adenosine receptor antagonists during preconditioning blocks the protective response 24 h later (Baxter et al, 1994) and stimulation of A l receptors results in marked protection from infarction 24-72 h later (Baxter and Yellon, 1997). Evidence is also present regarding N O as a key trigger for the preconditioning (Boll i et al, 1997). Based on the time course of delayed protection, the preconditioning may be involved in acquisition of 30 cytoprotective proteins, such as manganese-superoxide dismutase and heat shock protein 72 (Marber et al, 1993; Kuzuya et al, 1993). The detailed mechanism of delayed preconditioning remains to be explored. 1.3.4. Regulation of myocardial carbohydrate metabolism during postnatal development and ischemia. During postnatal development of the heart, myocardial glycogen content and metabolism of glucose and lactate decline as the dependence on fatty acids as an energy source increases (Hue et al, 1994; Veerkamp et al, 1985; Warshaw and Terry, 1970; Warshaw, 1972; Warshaw, 1970). Under normal conditions, the adult heart preferentially utilizes fatty acids while glycolysis is inhibited (glucose-sparing effect). Decreased fatty acid metabolism in the neonatal heart may reflect the delayed maturation of enzymes associated with mitochondrial fatty acid transport and metabolism, enzymes such as palmitoyl-CoA transferase, acetyl-CoA synthase and carnitine-palmitoyl transferase 1 (CPT1) (Warshaw and Terry, 1970; Warshaw, 1972; Warshaw, 1970). CPT1 is a key enzyme involved in transporting fatty acyl Co A into the mitochondria, facilitating fatty acid oxidation (Stanley et al, 1997; Fig. 4). During postnatal development of the heart, CPT1 activity is increased by a reduced cytosolic concentration of malonyl Co A which is a potent physiological inhibitor of CPT1 (Awan and Saggerson, 1993; Lopaschuk et al, 1994). Malonyl C o A is produced in the cytosol by the carboxylation of acetyl C o A , catalyzed by acetyl C o A carboxylase ( A C C ) . A C C , in turn, is negatively regulated by 5'-AMP-activated protein kinase ( A M P K ) (Witters and Kemp, 1992). Therefore, increased A M P K activity in the adult heart wil l result in an inhibition of A C C , a decrease of malonyl C o A concentration, an 31 increase of CPT1 activity and an acceleration of fatty acid oxidation. Insulin was shown to inactivate A M P K by a mechanism that still remains to be defined and decrease fatty acid oxidation (Makinde et al, 1997). Both the decreased circulating insulin levels and the increased expression of A M P K contribute to a higher activity of A M P K and a higher fatty acid oxidation in the adult than in the new born heart (Girard et al, 1992; Makinde et al, 1997). Concurrently to the decrease in fatty acid oxidation, enzyme activities involved in glycolysis also decrease during postnatal development of the heart (Veerkamp et al, 1985). The glucose-sparing effect of the adult heart has been proposed to result from an increase of citrate, which inhibits both 6-phosphofructo-l kinase (PFK-1) and 6-phosphofructo-2 kinase (PFK-2) (Hue et al, 1994). PFK-1 catalyzes the so-called 'first committed step' of glycolysis, while P F K - 2 is responsible for synthesis of fructose-2,6-biphosphate, which is a positive effector of PFK-1 (Hue et al, 1988). The activities of enzymes involved in the citric acid cycle are also low in the neonatal heart (Warshaw, 1972; Veerkamp et al, 1985). High glycogen content, a greater dependence on glycolysis, and the possible adaptive effects of fetal hypoxia on glucose 32 Figure 4. Putative role of insulin and other signalling pathways in the regulation of carbohydrate metabolism in the heart. The Solid arrows indicate the metabolic or signalling pathways. The blocked arrows indicate the inhibitory relationship where the increase or activation results in the inhibitory effect to the function of target protein. The dotted arrows show that insulin inhibits A M P K but the mechanism is still to be defined. For abbreviations, see the List of abbreviations. See pages 30-35 for more details of the relationships depicted in Figure 4. 33 Insulin receptor PI3K-PKB V PDK Glycogen DPG * y n t h a s e cAMPK i o Triacylglycerol Phosphorylase kinase Glycoge Glycogen ^ AMPK Malonyl-CoA phosphorylase \ . ^ ^ACC Fatty acids Fatty acyl-CoA ^ C P T , Fatty acylcarnitine Pyruvate < — > Lactate Acetyl-CoA \ Mitochondrial membrane \ Pyruvate dehydrogenase complex > ( Acetyl-CoA Citric acid cycle Fatty acy l -CoA<— Fatty acylcarnitine 3 4 metabolism may all contribute to the greater tolerance for hypoxia and ischemia of neonatal heart (Riva and Hearse, 1991; Murashita et al, 1992). In fact, during mild to moderate myocardial ischemia, increased translocation of glucose transporters (Glut-1 and Glut-4) and enhanced glycolysis both allow sustained A T P production to protect the ischemic heart (Lopaschuk and Stanley, 1997; Young et al. 1997; Neely and Morgan, 1974). Therefore, the metabolic changes, which take place during postnatal development, may underlie the increased susceptibility to ischemia of the adult heart. The rates of glycogen synthesis and breakdown are controlled respectively by glycogen synthase (GS) and glycogen phosphorylase (GP), which are influenced by both hormonal stimulation and metabolic feedback ( A M P , inorganic phosphate and Ca 2 + ) (Hue et al. 1994; Fig. 4). Stimulation of (3-adrenergic receptor increases glycogenolysis by activating glycogen phosphorylase. Glycogen phosphorylase is activated via phosphorylation by phosphorylase kinase, which is in turn activated by C a 2 + and c A P K (Alonso et al, 1995). During mild to moderate myocardial ischemia (20-60% reduction in coronary blood flow), the myocardial mechanical work is depressed as intracellular A T P concentrations decrease and the net output of lactate increases. In 30-90 min, A T P concentrations partially return to normal levels and lactate output decreases. However, the myocardial mechanical work remains depressed as long as the ischemia is present (Shultz et al., 1992). This contractile dysfunction under mild ischemia is called myocardial hibernation and acts to preserve the viability of the myocardium in response to decreased coronary oxygen delivery. When flow is restored, cardiac function returns to normal. During this mild ischemic period, cardiac oxygen consumption 35 decreases, anaerobic glycolysis (glycogen depletion and lactate accumulation) increases transiently and free fatty acid oxidation is reduced (Stanley et al, 1998). However, oxidative energy (ATP) still stems mainly from (3-oxidation of fatty acids (Liedtke, 1981). In a more severe ischemia (70% or more reduction of coronary blood flow), the rate of glycogen depletion and lactate accumulation is much greater (Guth et al, 1990; Marshall et al, 1981). As the source of fatty acids and glucose from circulation is greatly reduced, fatty acid oxidation is severely impaired and glycogen becomes the sole source of glycolytic substrate. Under normal condition, glycolysis is coupled to glucose oxidation and no H + is produced. However, under ischemic conditions, pyruvate derived from glycolysis is converted to lactate, yielding two H + from each glucose molecule (Opie, 1991). The production of H + is the major contributor to the acidosis that occurs in the myocardium during ischemia (Dennis et al, 1991). During reperfusion of the ischemic heart, intracellular p H recovers rapidly by an increase in sarcolemmal N a + - H + exchange and subsequent increase in intracellular N a + , which in turn activates the sarcolemmal N a + - C a 2 + exchanger. The increase of N a + - C a 2 + exchange rate may ultimately lead to C a 2 + overload and cell death (Karmazyn and Moffat, 1993). In addition to acidosis during ischemia, an overshoot in the rate of fatty acid oxidation and impaired pyruvate oxidation during reperfusion can further increase H + production and exacerbate injury (Lopaschuk et al, 1993). The mechanism for impaired glucose oxidation is unclear, but Kudo et al. (1995) demonstrated that intracellular malonyl C o A levels decrease to 40% of normal level during 30 min of total ischemia. The decrease of malonyl C o A levels appears to be due to increase in inhibition of acetyl C o A carboxylase by A M P K (Kudo et al, 1995). A t the same time, decreased levels of malonyl C o A wil l facilitate CPT-1 activity and fatty acid oxidation. Consequently, the high rates of fatty acid oxidation wil l result in inhibition of glucose oxidation (Hansford and Cohen, 1978). 3 6 During a brief ischemic preconditioning, glycogen contents are depleted and both lactate and H + production increase. The accumulated catabolites are then washed out by following reperfusion (Van Wylen, 1994), but the glycogen contents remain substantially depleted (Wolfe et al, 1993). When the glycogen-depleted heart is exposed to a prolonged ischemia, lactate and H + production are reduced and the depleted glycogen contents were timely correlated with the duration of the ischemic preconditioning (Wolfe et al, 1993). 1.3.5. Insulin mediated signalling pathways in the regulation of myocardial carbohydrate metabolism and protein synthesis. Upon binding to and activating its tyrosine kinase receptor, insulin stimulates multiple signalling pathways to increase glucose uptake, as well as synthesis of glycogen, lipid and protein (Denton and Tavare, 1995). The activated insulin receptor phosphorylates insulin receptor substrate-I (IRS-I), which subsequently recruits diverse signalling cascades. One such signalling pathway, which leads to enhanced protein synthesis, involves activation of phosphatidylinositol 3-kinases (PI3Ks), phosphatidylinositol 3,4-bisphosphate-dependent kinases (PDKs), protein kinase B (PKB) , F R A P , and the 70-kDa ribosomal protein S6 kinase (S6K) (Banerjee et al, 1990; Chung et al, 1992; Bos, 1995; Chou and Blenis, 1995; Downward, 1995; Franke et al, 1995; Alessi et al, 1997). The interaction between tyrosine-phosphorylated IRS-I and P D K s is mediated by the SH2 domain of P D K s . P D K s are a family of enzymes that phosphorylate inositol rings at the 3-position, generating phosphatidylinositol-(3,4)-phosphate (PtdIns(3,4)P2) or PtdIns(3,4,5)P3 (Toker and Cantley, 1997). PtdIns(3,4)P 2 and PtdIns(3,4,5)P3 can directly interact with P K B through the N-terminal pleckstrin homology (PH) domain of P K B and expose the Thr-308 and Ser-473 residues for phosphorylations (Alessi et al, 1996; 37 Alessi etal., 1997; Downward, 1998). The phosphorylations of these sites, mediated by P D K 1 and P D K 2 , respectively, render the enzyme fully active (Stephens et al, 1998). This activation of P K B leads to the phosphorylation and inhibition of glycogen synthase kinase (GSK) 3, which contributes to the stimulation of glycogen synthesis (Cross et al., 1995; Fig. 4). S6K, which participates in the regulation of m R N A translation, also requires phosphorylation of several residues by various protein kinases for its activation (Pullen et al, 1998; Downward, 1998). Even though, the detailed mechanism for the activation of S 6 K is still to be investigated, P D K 1 phosphorylates Thr-229 and activates S 6 K (Pullen et al, 1998). Another insulin signalling pathway has been proposed to activate the 85-kDa ribosomal S6 kinase (RSK) . This is initiated by binding of Grb2 via its SH2 domain to tyrosine-phosphorylated IRS-1. SOS, which is bound to Grb2, activates Ras, and this recruits Raf l to the plasma membrane where it becomes activated by phosphorylation. In a protein kinase cascade, the sequential activations of R a f l , M e k l and Mek2, E r k l and Erk2, and R s k l and Rsk2 have been proposed. Insulin signalling may also involve the activation of protein kinase C, either through pertussis toxin-sensitive trimeric G protein- or PI3K-dependent pathways (White, 1996; Taylor et al. 1995). The G protein-mediated pathway appears to be involved in activation of phosphatidylinositol-specific phospholipase C and/or D , whereas the PI3K-dependent pathway, may be involved in activation of phosphatidylcholine-specific phospholipase C and/or D . While much is known about the pathways that result in production of diacylglycerol, which activates P K C , the downstream actions of P K C in the context of insulin signalling are poorly defined. Likewise, insulin can induce activation of casein kinases (CK) 1 and 2 through obscure mechanisms (Allende and Allende, 1995). 38 The insulin enhancement of glycogen synthesis stems from activation of glycogen synthase. This enzyme is activated by dephosphorylation by protein phosphatase-1 (PP-1) and the inhibition of G S K 3 , which is mediated by P K B (Burgering and Coffer, 1995; Cross et al. 1995; Fig. 4). Insulin also increases glucose uptake into cells by translocating glucose transporters, mainly Glut-4, from an intracellular location to the plasma membrane (Denton and Tavare, 1995). Insulin-stimulated Glut-4 redistribution is suppressed by wortmannin and LY294002, specific P I3K inhibitors, but not by rapamycin (Denton and Tavare, 1995). This implies that the signalling pathway for Glut-4 translocation requires P I3K but not S6K. B y mechanisms that remain poorly defined, mitochondrial pyruvate dehydrogenase complex and cytosolic A C C , which participates in the conversion of pyruvate to fatty acid and the synthesis of malonyl-CoA, respectively, become activated by insulin. As discussed previously, insulin can inhibit A M P K , activate A C C and inhibit C P T 1 , resulting in the decrease of fatty acid oxidation (Fig. 4). 1.3.6. Histidine protein kinases and the two-component regulatory system. Since the first pioneering studies regarding reversible protein phosphorylation were performed by Edwin G. Krebs and Edmond H . Fisher, extensive efforts have made great advances in understanding the role of protein kinases in many cells. These studies have mainly focused on the phosphorylation of serine, threonine and tyrosine residues of target proteins, forming a phosphate ester link (O-phosphafe), which are carried out by enzymes, referred to as "O-kinases". In addition to the hydroxyl group phosphorylations, the nitrogen group can also be phosphorylated in histidine, arginine and lysine residues of proteins that are catalyzed by a family of protein called "N-kinases", forming a phosphoramidate bond (N-phosphate). The O-kinases are commonly found in eukaryotes, while the N-kinases predominant in prokaryotes. This phylogenetic dichotomy, however, has been contested by 39 the discovery of eukaryotic-like protein serine/threonine kinases and phosphatases in prokaryotes and bacterial histidine protein kinases in eukaryotic organisms (Kennelly and Potts, 1996). To date, histidine protein kinases have been identified in several lower eukaryotes including Saccharomyces cerevisiae, Arabidopsis thaliana, Neurospora crassa, and Dictyostelium discoideum (Kakimoto, 1996; Huang et al, 1991; Posas et al, 1996; Appleby et al, 1996). In mammals, no histidine protein kinase has yet been cloned, but the observations of histidine phosphorylations and histidine phosphatase activities imply its presence (Ohmori et al, 1993; Ohmori et al, 1994; Motojima and Goto, 1994; Crovello et al, 1995). The possible phosphoramidate bonds through nitrogen of arginine or lysine have been sporadically reported with little information for their regulation and roles (Matthews, 1995). 1.3.6.1. Chemical characteristics of phosphohistidine Unlike the phosphoester bond that occurs in the phosphorylation of serine, threonine or tyrosine, the phosphoramidate bond is unstable at acidic pH and this has caused technical difficulties in its detection and analysis. Many biochemical procedures involve acidic conditions such as protein precipitation by trichloroacetic acid, fixation of polyacrylamide gels in acetic acid, high performance liquid chromatography with trifluoroacetic acid or during Edman degradation and thus are not suitable for analysis of histidine phosphorylation. B y contrast, the phosphoramidate bond is stable at alkali pH, which allows phosphoamino acid analysis in alkaline hydrolyzates of proteins. In this condition, phosphoserine and phosphothreonine are lost. Early studies based on acid-lability have indicated the presence of histidine protein kinases in mammals (Walinder, 1969; Chen et al, 1974). Quantitatively, phosphohistidine was detected in about 6% of basic nuclear proteins from slime mold, Physarum polycephalum (Pesis, 1987). 40 The phosphoramidate bond in phosphohistidine can exist as one of two species (1-phosphohistidine and 3-phosphohistidine), depending on the nitrogen of the imidazole ring of histidine. In vitro, both of the 1- and 3-phosphohistidines are hydrolyzed at pH 2.4 and 46 °C with first-order rate constants of 0.2 and 0.4 min"1, respectively. These rate constants are greatly increased by either pyridine or hydroxylamine (Weigel et al, 1982). However, under biological conditions, it has been suggested that 1-phosphohistidine is more stable than 3-phosphohistidine (Hultquist et al, 1968), probably due to a peptide bond. Interestingly, most of the phosphohistidine from rat liver extracts is 3-phosphohistidine, whereas in yeast and Escherichia coli, 1-phosphohistidine is the main form (Walinder, 1969). 1.3.6.2. The two-component regulatory system in prokaryotes. The basic paradigm for the two-component system involves five protein domains on two proteins, the sensor kinase and response regulator (Matthews, 1995; Appley et al, 1996, Kennelly and Potts, 1996). The sensor kinase contains an extracellular input domain (receptor) at the N-terminal region and an intracellular transmitter domain composed of a kinase domain (an autophosphorylating protein-histidine kinase) and a substrate domain at C-terminus. The kinase and substrate domains span about 240 amino acids with several amino acid motifs which are conserved in most histidine protein kinases. One of these, termed the H-box, contains a putative autophosphorylation site within a H y d - S - H * - X - X - R - X - X - L motif, where Hyd represent a hydrophobic residue and X for any residues. Other boxes include nucleotide binding glycine-rich domains, G I and G2, and F and N boxes with undefined functions (Kakimoto, 1996). The response regulator usually contains a receiver domain (approximately 120 amino acids in length at N-terminus), which catalyzes transfer of the phosphate group from the histidine to a 41 conserved aspartate residue and an output domain, commonly a transcriptional regulator (Appleby, 1996). This paradigm can be illustrated by the Escherichia coli osmosensing signal transduction pathway, which comprises EnvZ and OmpR (Fig. 2C). The EnvZ is a sensor kinase that detects the changes of extracellular osmolarity by an unknown mechanism and transfers a phosphate to the response regulator, OmpR, after autophosphorylating a histidine residue. The autophosphorylation is believed to be a trans-phosphorylation in a homodimerized EnvZ (Wurgler-Murphy, 1997). The phosphorylation state of OmpR regulates the expression of OmpF and OmpC genes which encode porin proteins of the outer membrane. In typical two-component regulatory systems, the phosphorelay between the sensor kinase, which contains the first autophosphorylating histidine site (HI) and the response regulator with phosphate-receiving Asp residue (D2) is mediated by other proteins containing phosphorylating His (H2) and Asp (DI) but lacking kinase domains. In prokaryotes, it has been estimated that 40-50 two-component regulatory systems are involved in regulating transcription and cell behavior in Escherichia coli (Stock et ai, 1989). The well-studied systems include CheA/CheY for chemotaxis, NRn /NRi for nitrogen metabolism and PhoR/PhoB for phosphate metabolism (Stock etal, 1989). Within the two-component regulatory system, there are subclasses that can be grouped, based on the number of proteins involved. The first group involves a chain of four proteins, which can be exemplified in the signalling pathway for the initiation of sporulation in Bacillus subtilis (Burbulys et al, 1991). The signal initiates with activation of one of three sensor kinases, K i n A , K inB or KinC. These contain a receptor and an autophosphorylating histidine 42 kinase (HI) (Fig. 2C). The phosphoryl group is transferred to an aspartate group of SpoOF ( D l ) , a receiver protein, and is then transferred to an aspartate group of the transcription regulator, SpoOA (D2), via another in histidine of SpoOB (H2). The second subgroup of the two-component regulatory system comprises two proteins where the sensor kinase has integrated the first three steps (H1-D1-H2). This subgroup has been discovered in several microorganisms including BvgS of Bordetella pertussis which is involved in the synthesis of toxins (Uhl and Miller, 1996), FrzE of Myxococcus xanthus involved in chemotaxis (McCleary and Zusman, 1990), V i r A of Agrobacterium tumefaciens involved in host plant recognition (Jin et al., 1990), and A r c B of E. coli involved in recognition of anaerobic environment (Iuchi, 1993). The third subgroup comprises three phosphorelaying proteins where the first two steps of phosphorylation are fused into a sensor receptor protein ( H l - D l ) . The best known example for this group is found in the osmoregulation response of the budding yeast, Saccharomyces cerevisiae (Posas et al, 1996). S ln l is a transmembrane osmosensory protein containing an extracellular sensor domain, a cytoplasmic histidine kinase and a receiver domain. At low osmolarity, the S ln l is activated and autophosphorylates at His-576. Subsequently, the phosphate group is transferred to Asp-1144 ( H l - D l ) . The phosphate group of Asp-1144 is then transferred to Asp-554 of Ssk l (D2) via phosphorylation of another protein, Y p d l , at His-64 (H2) (detailed mechanism discussed in next section). Besides the aforementioned subgroups, several phosphorelaying architectures have been postulated and recognized (Appleby et al, 1996). These combinations of architectures include H 1 ^ D 1 - H 2 - D 2 , H1->D1-H2->D2, H 1 - > D 1 ^ H 2 - D 2 or H1-D1-H2-D2. At the same time, the phosphorelay may not necessarily be linear, but could be branched. In E. coli, for example, the A r c B / A r c A pathway displays both HI—>D2 and H2—»D2 phosphorelaying pathways (Tsuzuki et 43 al, 1995). 1.3.6.3. Lower eukaryotic two-component regulatory system involved in Hogl MAP kinase pathway. The two-component regulatory system has been identified in several lower eukaryotes. In budding yeast (Saccharomyces cerevisiae), the H O G signalling cascade plays a key role in the adaptation of cells to high osmolarity. The activation of H o g l induces transcription of several genes including NAD-dependent glycerol-3-phosphate dehydrogenase (GPD), which is important for the glycerol synthesis and expression of stress response genes such as catalase T and heat shock protein 12 (Schuller et al., 1994; Varela et al., 1995). This signalling pathway is regulated by two sensory inputs, the Shol transmembrane protein that contains an SH3 domain and the two-component regulatory system (Slnl) (Maeda et al, 1995). At normal osmolarity, the S ln l is activated and keeps S s k l phosphorylated by the phosphorelay mechanism discussed in the previous section. The phosphorylated Ssk l inhibits activation of Ssk2 and Ssk22. At high osmolarity, the S ln l is inactivated, resulting in the accumulation of dephosphorylated S s k l , which allows the activation of Ssk2 and Ssk22. The dephosphorylated S s k l interacts with an inhibitory N-terminal segment of Ssk2 via C-terminal receiver domain and activates Ssk2 (Maeda et al, 1995). Activated Ssk2 autophosphorylates by an intramolecular reaction and, once phosphorylated, Ssk2 does not require Ssk l for its catalytic activity (Posas and Saito, 1998). These observations are similar to M e k k l where autophosphorylation is crucial for the activation of M e k k l (Siow et al, 1997). M e k k l autophosphorylation also results from an intramolecular reaction (Deak and Templeton, 1997). Activated Ssk2 then phosphorylates and activates Pbs2, a M A P K K , which is an upstream kinase for H o g l . A similar two-step mechanism 44 may be found in mammalian stress-induced signal transduction pathways. A homolog of Ssk2/Ssk22 was identified, named M T K 1 and shown to be involved in p38 and J N K signalling pathways (Takekawa et al, 1997). The mouse homolog of M T K 1 , known as Mekk4, binds to Rac and Cdc42, and is involved in the activation of J N K signalling pathways (Gerwins et al, 1997). However, it still not yet known whether the regulator of M T K 1 is structurally related to S s k l . In fission yeast {Schizosaccharomyces pombe), W i s l ( M E K ) and S ty l ( M A P kinase), which are homologous to Pbs2 and H O G 1 , respectively, have been identified. These kinases are required for cell cycle, initiation of sexual differentiation and protection against cellular stresses (Shiozaki and Russell, 1995). A wide range of environmental stresses, including osmotic stress, oxidative stress, D N A damaging agents, heat shock and the protein synthesis inhibitor anisomycin, activate these kinases. The activation of W i s l and S ty l is regulated by Wis activating kinasel (Wakl) and mitotic catastrophe suppressor (Mcs4), which are structurally and functionally homologous to the budding yeast Ssk2/Ssk22 M E K kinase and S s k l response regulator, respectively (Shieh et al, 1997). Even though the sensor histidine kinase is still to be identified, it appears that a two-component regulatory system also controls the S ty l activity. Genes encoding two-component regulatory proteins have also been identified in the filamentous fungus, Neurospora crassa, and the plant, Arabidopsis. In N. crassa, Nik-1 encodes a protein that shares homology with both the histidine kinase and regulator module of the two-component regulatory system and appears to be involved in hyphal development (Alex et al, 1996). E T R 1 , an ethylene receptor, and CKI1 (cytokinin-independent-1), a possible cytokinin 45 receptor of A. thaliana, also contains the sensory histidine kinase and regulator domains (Kakimoto, 1996; Schaller and Bleecker, 1995). The signalling pathway of E T R 1 is similar to S ln l in yeast in that the E T R 1 histidine kinase is active in the absence of ethylene and negatively regulates the down stream element, CTR1 which is a Raf family protein kinase (Kieber et al, 1993; Schaller and Bleecker, 1995). A noteworthy aspect of eukaryotic two-component regulatory systems is that, unlike those in prokaryotes that have both sensory receptor and effector functions such as transcriptional activation, they represent the initial segment of more extensive signalling cascades. In addition to the transmembrane receptor histidine kinases, other histidine protein kinases, such as N i k l of the fungus Neurospora and D o k A of the slime mold Dictyostelium, lack the transmembrane segment. Little is known about these kinases except that they are implicated in the response of osmolarity changes and perhaps, are regulated by other transmembrane osmosensor proteins (Alex et al, 1996). 1.3.6.4. Histidine kinases in mammalian systems. Even though the presence of phosphohistidines in mammalian systems has been known for about 30 years, no histidine protein kinase has yet been cloned. However, the identification of a histidine protein kinase and possible regulating signalling pathways have been reported in rat liver extracts and human platelets. A phosphohistidine containing protein with a molecular mass of a 38-kDa has been found in rat liver membrane extracts (Hedge and Das, 1987; Matthews et al, 1993). The phosphorylation of a 38-kDa protein was enhanced in the presence of activated small G protein, Ras, and glucagon in vivo and in vitro (Hedge and Das, 1990). Separate from these studies, Motojima and Goto (1993, 1994) have observed histidyl phosphorylations of a 36-kDa protein induced by the peroxisome proliferator, clofibrate. The level of expression of the 4 6 38-kDa protein was significantly increased during development from fetus to adult in both rat and human liver (Hedge, et al, 1993) and was persistently phosphorylated in hepatoma Fao cells (Motojima et al, 1994). The 36-kDa and 38-kDa proteins appear to be identical and have been identified as ornithine transcarbamylase (OTC) (Matthews, 1995). To date, the role of the histidyl phosphorylation and the responsible kinase have not been identified. Another phosphohistidine was found in p-Selectin, a leukocyte adhesion molecule, on the C-terminal cytoplasmic tail, in response to thrombin and collagen in human platelets (Crovello et al, 1995). The responsible histidine kinase still remains elusive. 47 2. HYPOTHESIS Protein kinases are crucial components relaying signals from specific extracellular stimuli to intracellular targets. A distinct pattern of these signalling pathways is activated by an extracellular stimulus, which will result in a specific physiological effect. During postnatal development of the heart, these protein kinases are regulated both in expression and activity. These changes are important to allow the heart to cope with various functions required during development and are also implicated in age-specific responses to various stimuli. In the heart, the signalling pathways for mitogenesis, stress-responses and glucose metabolism are differentially regulated and this could in part account for terminal differentiation of cardiomyocytes, less tolerance to hypoxia or ischemia and decreased dependence on the glucose metabolism for energy production in adult hearts. 48 3. Objectives. Among the several signal transduction cascades known to date, M A P kinase related-cascades are involved in the regulation of cell proliferation/differentiation, stress-responses and apoptosis. Distinct signalling cascades involving PI3K and P K B have been implicated in the regulation of glucose metabolism and protein synthesis. The family of C D K s serves as regulators of cell cycle progression and proliferation. The cyclic nucleotide-dependent protein kinases, P K A and P K G , and second messenger-regulated protein kinases, P K C and C a M P K , are important in cardiac contractility and cell growth. The purpose of this study was to investigate how these signalling pathways are regulated by extracellular stimuli and to study the developmental changes in their expression and activities. Furthermore, potentially novel protein kinases involved in the development of the rat heart were investigated. Specific aims for this study 1. To determine the detailed expression of various known protein kinases involved in the signal pathways in postnatal development. 2. To determine the activities of these protein kinases during the postnatal development of rat heart ventricle. 3. To investigate the regulation of protein kinases by exogenous stimuli. 4. To identify potentially novel protein kinases involved in postnatal development. 5. To purify and characterize a histidine protein kinase present in adult bovine heart. 49 4. M E T H O D S A N D M A T E R I A L S 4.1. Materials A l l chemical reagents were purchased from either B D H (Vancouver, B .C . , Canada), S I G M A Chemical Co. (St. Louis, Mo. , U S A ) or FISHER Scientific Co. (New Jersey, U S A ) . For rat tissue preparations, sodium pentobarbital (Somnotol®) and heparin sulfate were purchased from M T C Pharmaceuticals (Cambridge, Ont., Canada) and Organon Technika Inc. (Toronto, Ont., Canada), respectively. For isolation of ventricular myocytes, Joklik-modified minimal essential medium (powder) and collagenase were obtained from G I B C O Life Technologies (Grand Island, N Y , USA) and Worthington Biochemical Corp. (Freehold, NJ , U S A ) , respectively. Primary antibodies for Western blotting were obtained from Kinetek Pharmaceutical, Inc. (Vancouver, B .C . , Canada), Santa Cruz Biotechnology (Santa Cruz, C A . , U S A ) , Transduction Laboratory (Mississauga, Ont., Canada) or Upstate Biotechnology Inc. (Lake Placid, N . Y . , U S A ) as indicated in Table 1. The secondary antibodies for goat anti-rabbit IgG of alkaline phosphatase conjugate and horseradish peroxidase conjugate were obtained from Bio-Rad (Hercules, C A . , U S A ) and Calbiochem (San Diego, C A . , U S A ) , respectively. Enhanced chemiluminescence (ECL) detection reagents for immunoblotting were purchased from Amersham (Oakville, Ont., Canada). Various substrates for kinases were obtained from Kinetek Pharmaceutical, Inc. (Vancouver, B.C. , Canada) unless stated otherwise. 8-Br-cGMP, forskolin, 8-CPT-cAMP, P M A , PD98059 and SB203580 were purchased from Calbiochem (San Diego, C A . , USA) . [y- 3 2 P]ATP was obtained fromDuPont (Washington, D C , USA) . 50 Table 1. Antibodies and amino acid sequences of synthetic peptides used to raise antibodies. Antibodies were obtained from Kinetek Pharmaceutical Inc. (Vancouver B C , Canadada) or purchased commercially. For antibodies from Kinetek Pharmaceutical Inc., the peptides in P B S and Freund's incomplete adjuvant were injected into rabbits and serum was obtained, after several boosting injections. The antibodies were purified using peptide affinity columns and titred by E L I S A . The species in which antibodies were raised are indicated in the "Origin" column. For commercially purchased antibodies, the peptides are indicated for the corresponding kinase residues with their species of origins and the companies from which the antibody was purchased: SC, Santa Cruz Biotechnology (Santa Cruz, C A ) ; T L , Transduction Laboratory (Mississauga O N , Canada); U B I , Upstate Biotechnology Inc. (Lake Placid, N Y ) . Kinase Immunogen amino acid sequence or residues Origin Source CaMPK II N T - C T R F T D E Y Q L F E E L P C T - E E T R V W H R R D G K W Q N V H F H C Rat Rat Kinetek Kinetek C A P K N T - K K G S E Q E S V K E F L A K C Human Kinetek C D K 1 IX-PLFHDS EIDQLFRIFR A L G T P - G G C CT-CLS K M L V YDPAKRIS G K M A L K H P Y F D D L D N Q I K K M Mouse Mouse Kinetek Kinetek CDK2 M2-residues 283-298 (C-terminus) Human SC CDK3 Y20-residues 286-304 (C-terminus) Human SC CDK4 H22-residues 282-303 (C-terminus) Human SC CDK5 CT-CNPVQRISAEEALQHP Mouse Kinetek CDK7 P C T - V A T K R K R A E A L E Q G C Mouse Kinetek CDK8 N T - M D Y D F K V K L S S E R E R C Human Kinetek 51 C K 2 III-LKPVKKKKIKREIKILENLR-GGC Human UBI Erk 1 C T - C G G P F T F D M E L D D L P K E R L K E L I F Q E T A R F Q P G A P E A P Rat UBI Erk2 C14-residues 345-358 Rat SC Gsk3p XI-CSHSFFDELRDDPNNK Rat Kinetek K K C T - C L D N K K Y Y S D T K K L N Y R C13-residues 346-358 Human Kinetek SC Mapkapk2 PCT-S R V L K E D K E R W E D V K G C Mouse UBI Mekl X I - E F Q D F V N K C L V K N P A E R A D L K C NT-MPKKKPTPIQLNC Mouse Mouse Kinetek Kinetek Mekkl N T - C G S T H F T R M R R R L M A I A D Mouse Kinetek M K K 3 C T - K T K K T D I A A F VKILGEDS C Human UBI Mlk3 C20- residues 828-847 Human SC Mos III-ASQRSFWAELNIARLRHDNIVRAWAASTR M20-residues 371-390 Frog Mouse Kinetek SC p38 Hog C20-residues 341-360 NT-MS QERPTF Y R Q E K C Mouse Mouse SC UBI Pak 1 N T - M S N N G L D V Q D K P C N20- residue 2-21 Mouse Rat Kinetek SC Pak2 NT- M E E T Q Q K S N L E L L S A C Human Kinetek Pak 3 NT-MSDSLDNEEKPPAC Rat Kinetek PI3K Recombinant p85 PI3K, monoclonal Human UBI cAPK N T - K K G S E Q E S V K E F L A K C Human Kinetek PKB P H - F H V E T P E E R E E V T C CT-CRRPHFPQFS YS AS ST A Human Human Kinetek UBI 52 PKC a M7-purified PKC, monoclonal Rabbit UBI PKC i Residues 404-587, monoclonal Human T L cGPK CT-CDEPPPDDNS GWDIDF Bull UBI cGPK l a N T - E L E E L F A K I L M L K E E L Bull UBI R a f i C20- residue 629-648 Human SC RafA CT-residues 584-597 Human UBI RafB CT-residues 619-632 Human UBI Rsk 1 C21 -residue 716-735 Human SC Rsk2 PCT-CNRNQSPVLEPVGRS Mouse UBI S6K CT-CFPMIS K R P E H L R M N L NT-AGVFDIDLDQPEDAGSEDELEEGGQLNESC Human Human UBI UBI SAPK a Whole protein Rat Kinetek S A P K p NT-MS KS K V D N Q F YS V E V G C Human SC JNK1 C17- residue 368-384 Human SC Sek 1 XI-CLTKDES K R P K Y D E L L K CT-CKILDQMPATPSSPMYVD Xenopus Mouse UBI Kinetek T A K 1 CT-CKKQLEVIRSQQQKRQGTS M17-residues 563-579 Mouse Mouse Kinetek SC Tpl-2 (Cot) NT-MEYMSTGSDNKEEIDC CT-RGHQVIHEGSSTNDPNNSC Rat Rat Kinetek Kinetek Weel X - L T V V C A A G A E P L P R N G D Q W H E I R Q G R L P - C G G T-NTS S HR Y G L R R G D Q M M E D W Q V N V - G G C Human S. pombe Kinetek Kinetek 53 4.2. Animals Male Sprague-Dawley rats (1- to 365-day old) were obtained from the Animal Care Facility of the University of British Columbia. Two to three rats were caged with access to water and food ad lib. Bovine hearts were obtained from Olympia Fine Meats (Vancouver) and transported to the laboratory on ice. 4.3. Methods 4.3.1. Preparation of various rat tissues Hearts from 1-, 10-, 20-, 50-day and 1 year old male Sprague-Dawley rats were rapidly excised, after induction of anesthesia by intraperitoneal injection of sodium pentobarbital (60 mg/Kg) and heparin (2 U/g). The ventricle of the heart was rinsed with phosphate-buffered saline at 4 °C, frozen in liquid nitrogen, and stored at -70 °C until use. The ventricular tissue was pulvurized with 5 strokes of a liquid nitrogen-cooled hand French press. The pulverized tissue was then resuspended in 10 volumes of ice-cold homogenization buffer, containing 20 m M M O P S , 15 m M E G T A , 2 m M N a 2 E D T A , 1 m M N a 3 V 0 4 , 1 m M dithiothreitol (DTT), 75 m M (3-glycerophosphate, 0.1 m M P M S F , 1 ug/ml aprotinin, 10 ug/ml pepstatin A , and 1 ug/ml leupeptin and sonicated with a Branson Probe Sonicator at 4°C with three bursts of 30 s each at an output of 4%, duty of 80, and output control setting at 2.5. The resulting homogenate was centrifuged at 100,000 rpm (240,000 x g) for 11 min in a Beckman TLA-100.2 ultracentrifuge. The supernatant was used as the cytosolic fraction. The pellet was washed with the homogenization buffer and centrifuged as before. The second pellets were resuspended in the homogenization buffer containing 1% Triton X-100 (Membrane Grade) and sonicated as before and incubated for 15 min on ice, followed by centrifugation as before. This final supernatant was saved as the detergent-solubilized particulate fraction. Other rat tissues including 54 adipose, brain, intestine, kidney, spleen, testis, skeletal muscle (hind leg tibial muscle) and thymus were also collected from 50 day-old rats and homogenized as before with a homogenization buffer containing 1 % Triton X-100. 4.3.2. Preparation of isolated rat ventricular myocytes. The isolation procedure was modified from the protocol of Rodrigues et al. (1997). Fifty-day old rat hearts were excised as described above and each heart was perfused in retrograde mode with Buffer A (Joklik minimal essential medium, containing 2 m M NaHCCb, 1.2 m M MgSCM and 1 m M DL-carnitine) for 5 min, followed by the same buffer containing 25 u M C a 2 + and 75 mU/ml of collagenase (Type II, Worthington) for 30 min at 37 °C. The softened ventricular tissue was then cut and teased to small pieces with a scalpel, and incubated for 10 min in the same collagenase and C a 2 + containing Buffer A with occasional agitation. Dissociated ventricular myocytes were carefully aspirated and passed through a 200 pm mesh silk screen to remove fine tissue debris. The isolated ventricular myocyctes were then sequentially re-suspended in Buffer A containing 50 u M , 100 u M , 500 u M and 1 m M C a z + . The cells were then pelleted by centrifugation for 60 s at 45 x g. The cell pellet was carefully loaded onto Buffer A containing 1 m M CaCb. and 4% B S A to separate viable cells from dead cells. Rod-shaped viable cells are larger and asymmetric, and therefore, settled more quickly than non-viable cells. After the settling for 10 min, the supernatant was discarded and the cells were re-suspended into Buffer A containing 1 m M CaCk. The isolated ventricular myocytes were then sonicated with three bursts of 30s each on ice-cold homogenization buffer and the homogenates were then fractionated with cytosolic- and detergent- soluble fractions as described in the previous section. 55 4.3.3. Stimulation of ventricular tissues and cardiomyocytes. 4.3.3.1 In vivo treatment of insulin by tail vein injection. For insulin treatment, 50-day old rats were fasted overnight and insulin (2 U/kg) was injected into the tail vein 2 or 5 min before excision of the hearts. Control rats were injected with saline instead of insulin. The ventricles of the hearts were removed, rinsed with phosphate buffered saline at 4 °C, frozen in liquid nitrogen, and stored at -70 °C until use. The ventricular tissues were pulverized with 5 strokes of a liquid nitrogen-cooled hand French Press and re-suspended in 10 volumes of ice-cold homogenization buffer, containing: 20 m M M O P S , 15 m M E G T A , 2 m M N a i E D T A , 1 m M N a 3 V 0 4 , 1 m M DTT, 75 m M 6-glycerophosphate, 0.1 m M P M S F , 1 ug/ml aprotinin, 0.7 ug/ml pepstatin, and 1 ug/ml leupeptin, and sonicated with a Branson Probe Sonicator at 4 °C with 3 x 30 s bursts. 4.3.3.2. Preparation in Langendorff-perfused isolated rat hearts. After administration of heparin (2 U/g) and sodium pentobarbital (80 mg/kg) by intraperitoneal injection, hearts were excised from 250-300 g rats and attached by the aorta to the Langendorff-perfusion apparatus and perfused via a peristaltic pump in a retrograde flow with the Buffer A containing 1 m M CaCb. for 5 min without recirculating the buffer at 10 mVmin. After extraneous tissues and blood were removed, various agonists including 20 u M adenosine, 50 ng/ml anisomycin and 0.5 M sorbitol were added to the 50 ml of buffer and the buffer was recirculated until the heart was fast frozen in liquid nitrogen. For control hearts, the buffer was recirculated for 5 min without adding any agonists. The frozen hearts were pulverized with 5 strokes of a liquid nitrogen-cooled hand French press. The pulverized tissue was then suspended in 10 volume of ice-cold homogenization buffered and sonicated with a Branson Probe Sonicator at 4 °C with three bursts of 56 30 s each at an output of 4, % duty of 80, and output control setting at 2.5. The resulting homogenate was centrifuged and fractionated as described in Section 4.3.1. 4.3.3.3. Treatment of isolated adult ventricular myocytes with various agents. Isolated ventricular myocytes, prepared as described in Section 4.3.2, were stabilized in Buffer A containing 1 m M CaCk for 1 h and microscopically checked for viability and cell number. The cell suspension was then divided into aliquots of 0.5-1 million viable cells and treated with various agents indicated in the figures for a given time. The reactions were stopped by freezing the cells in liquid nitrogen after centrifuging the cell suspension for 30 s at 100 x g and aspirating the supernatant. The frozen cell pellets were thawed in 1 ml of homogenization buffer containing 1% triton-X 100 and cells homogenized by three burst of 10 s sonication, yielding 1-2 mg/ml of proteins. The homogenates were centrifuged and total cell lysates were obtained as described in Section 4.3.1. 4.3.4. Determination of protein concentration. Protein concentration was determined by the dye binding method (Bradford, 1976), using the reagents and procedures for the micro-assay supplied by Bio-Rad Laboratories. The linear standard curve was prepared by reading the absorbance of 0 to 25 ug of B S A at 595 nm emission wavelength. 4.3.5. Column chromatography. 4.3.5.1. Ion-exchange column chromatography. Anion- or cation-exchange chromatography was carried out with 1-ml Mono Q, Mono S, Resource S or Resource Q HR5/5 columns attached to a F P L C system (Pharmacia). Crude extracts 57 (1-5 mg of protein) were loaded onto the column which was previously equilibrated with Mono Q buffer (10 m M M O P S (pH7.2), 5 m M E G T A , 2 m M E D T A , 1 m M N a 3 V 0 4 , 1 m M D T T and 25 m M ^-glycerophosphate) or Mono S buffer (the same as Mono Q except p H 6.8). After washing off unbound proteins with 1 volume of Mono Q buffer, bound proteins were eluted with a 10-ml gradient of 0-0.8 M NaCl at a flow rate of 1 ml/min and 0.25-ml fractions were collected. The fractions assayed immediately after elution or stored at -70 °C until assayed. 4.3.5.2. Superose 12 gel-filtration chromatography. The 40-kDa histidine kinase was partially purified from adult ventricular extracts and a sample (200 ul) was loaded onto a Superose 12 (HR10/30) column that was equilibrated with 0.25 M NaCl in KII buffer for 1 h (12.5 m M M O P S , 12.5 p-glycerophosphate, 0.5 m M E G T A , 7.5 m M M g C k and 0.05 m M NaF). Proteins were run through the column with 20 ml of the above buffer at a rate of 0.25 ml/min and 250 ul per fractions were collected. Each fraction was assayed for autophosphorylating histidine kinase activity. To calibrate the column, a mixture of standards (1 mg each of 67-kDa bovine serum albumin, 43-kDa ovalbumin and 25-kDa chymotrypsinogen) was loaded and eluted with the same protocol. The sample fractions were assayed for protein concentrations by using a Bradford protein assay system as described in Section 4.3.4. 4.3.6. SDS-polyacrylamide gel electrophoresis, transblotting and immunoblotting. The protein concentrations of samples were assayed with Bradford reagent and then adjusted by diluting with 1% SDS to yield identical protein concentrations. Electrophoresis was performed in S D S - P A G E , using the discontinuous buffer system (Laemmli, 1970). Proteins were subjected to electrophoresis on 1.5 mm thick polyacrylamide gels, with a stacking gel containing 4% acrylamide 58 and a separating gel containing 10 to 12.5% acrylamide. Before loading, the samples (except boiling labile samples) were boiled for 3 min with Sample loading buffer (2.5% SDS, 10 % glycerol, 50 m M H Q , pH 6.8, 0.5 M fj-mercaptoethanol and 0.01% bromophenol blue), then briefly centrifuged. The gels were subjected to electrophoresis at 8 to 23 mA until the dye front reached the bottom of the gel. After dissembling the S D S - P A G E apparatus, the proteins were electrophoretically transferred onto nitrocellulose or P V D F membranes at 300 mA for 3 h. Membranes were then blocked with 3% skimmed milk powder in Tris-buffered saline (20 m M Tris/HCl, pH7.5, 0.5 M NaCl , 0.2 % Tween-20; TBST) for 15 min, quickly rinsed with TBST, and incubated for 2 h with primary antibody in T B S T with constant shaking at room temperature. The membrane was washed three times for 10 min with T B S T and incubated with horseradish peroxidase conjugated secondary antibody (goat anti-rabbit or anti-mouse IgG) in T B S T for 30 min. After washing the membrane three times for 10 min with TBST, immunoreactivity was visualized using the E C L Western blotting detection system (Amersham). For the colorimetric assay, alkaline phosphatase (ALP) conjugated secondary antibody (goat anti-rabbit or anti-mouse IgG) was used instead of using horseradish peroxidase conjugated secondary antibody. After rinsing the membrane briefly with 1 x A P buffer, color was developed with 50 ml A P buffer containing 340 pi of N B T (50 mg/ml in 70% D M F ) and 170 p i of BCIP (50 mg/ml in 100 D M F ) . The color reaction proceeded from 30 s to 3 h, depending on intensity of the bands and was terminated by rinsing the membrane with dH20. 4.3.7. Stripping of Western blot membrane. Occasionally, membranes were stripped and re-probed once or twice. For E C L blots, membrane was stripped with buffer containing 100 m M p-mecaptoethanol, 2% SDS and 62.5 m M Tris-HCl, p H 59 6.7 for 30 min at 60 °C with agitation every 10 min. For A L P blots, the membrane was incubated in T BS (pH 2.5) for 10 min, followed by two 5 min washes with TBS (pH 7.5). In either case, membranes were re-blocked with 5% skim milk powder or B S A in TBST. 4.3.8. Two-dimensional electrophoresis. Two-dimensional electrophoresis was performed by using isoelectric focusing (IEF) gel as the first dimension and S D S - P A G E as the second dimension. The first dimension was carried out by using immobilized p H gradient (IPG) gel systems. Immobiline DryStrip (Pharmacia), 110 mm or 180 mm with p H 3-10, was used as described in the provided instructions with slight modifications. Briefly, precast immobilized pH gradient polyacrylamide gel strips were re-hydrated with 250-400 ul of Re-hydration buffer (Pharmacia) over night in a re-swelling cassette. The re-hydrated strips were placed onto Multiphor II unit and covered with mineral oil. Samples (50-100 ul) were then loaded in the sample cups. IEF was performed at 300 V for 1 h, then 1400 V for 14-36 h. Following IEF, the strips were equilibrated with S D S - P A G E sample buffer for 30 min in the re-swelling cassette and loaded onto 11% S D S - P A G E gels. The strips were immobilized by using 0.4% agarose and usual S D S - P A G E was performed as described previously. 4.3.9. Gel and membrane staining. 4.3.9.1. Ponceau staining. Membranes were immersed in Ponceau S solution (0.1% w/v in 1% v/v acetic acid) for 30 s, followed by destaining in dH20 for 10 s. 4.3.9.2. Amido black staining. 60 Membranes were immersed in 0.1% amido black in 40% methanol for 10 to 20 min at room temperature with shaking. De-staining was carried out in a 5% methanol solution until bands were visible and background was diminished. 4.3.9.3. Silver staining for SDS-PAGE gels. After electrophoresis, gels were fixed by soaking in 200 ml of solution containing 10% acetic acid and 40% methanol for 30 min, followed by 10% ethanol and 5% acetic acid (v/v/v) for 15 min twice. The gels were washed twice in 10 min in excess of dH 20 to facilitate rehydration of the gels and to remove methanol. The gels were then oxidized for 5 min in oxidizer containing 3.4 m M KsCteO and 3.2 m M nitric acid, washed three times with dH.20, and stained with 0.2% AgNCb (w/v) for 20 min. After briefly washing the gels, a dark brown color was developed with 0.28 M Na2CC>3 in a formaldehyde solution (0.166% v/v). The color development was terminated by immersing the gels in 5% v/v of acetic acid. 4.3.9.4. Colloidal silver staining for membranes. Colloidal silver staining solution was prepared by adding 0.5 ml of 40% w/v sodium citrate and 0.4 ml of 20% (w/v) ferrous sulfate into 9 ml of d tkO. After voltexing the yellowish liquid, 0.1 ml of 20% (w/v) silver nitrate was added dropwise with vigorous vortexing to produce a dark-brown solution. Membranes were immersed in the stain until developed as desired (up to 10 min) and then destained with several washes of dH20. 4.3.10. Autoradiography and development of film. To visualize radiolabeled or fluorescent attached proteins, gels or membranes were wrapped in 61 Saran wrap to protect the film from moisture and then exposed to film. Typically the film was exposed for 10-60 s for E C L and 1-6 days for radiolabeled proteins. Films were developed in Kodak Developer for 30 s, followed by stopping in 3% acetic acid (v/v) for 60 s, and fixing in Kodak Fixer for 3 min. After washing the film in running water for 5 min, the film was air-dried. 4.3.11. Protein kinase assays. 4.3.11.1. Measurement of MBP, c-Jun and hsp27 phosphotransferase activities from crude extracts. Myelin basic protein (MBP), human recombinant heat shock protein 27 (hsp27), and GST-c-Jun (1-79) phosphotransferase activity was measured by adding 30 to 60 ug protein of crude extracts (30 ul) into Reaction buffer (25 m M p-glycerol phosphate, 20 m M M O P S , p H 7.2, 5 m M E G T A , 2 m M E D T A , 20 m M M g C l 2 , 1 m M Na V 0 4 , 0.25 m M DTT, and 5 m M p-methyl aspartic acid) containing either 10 ug, 1 ug, or 4 ug of M B P , hsp27, and GST-c-Jun, respectively, as a substrate. The reactions were started by addition of 50 p M [y- 3 2 P]ATP (2000 cpm/pmol). After incubating the reaction mixtures at 30 °C for 10 min, the reactions were terminated by adding 5 x Sample loading buffer. For GST-c-Jun assays, the reactions were stopped by adding ice-cold KII buffer (12.5 m M M O P S , 12.5 ^-glycerophosphate, 0.5 m M E G T A , 7.5 m M M g C h and 0.05 m M NaF) and the beads were washed three times with the same buffer. The phosphorylated proteins was resolved on 11% S D S - P A G E gels and transblotted onto nitrocellulose or P V D F membrane. The M B P , hsp27 and GST-c-Jun bands were visualized by Ponceau staining as well as autoradiography. The phosphotransferase activities were quantified by counting the excised bands in scintillation cocktail or by densitometry analysis of the phosphoimages. 62 4.3.11.2. Measurement of MBP, c-Jun, hsp27, S6-10, casein phosphotransferase activities from column fractionated extracts. M B P , GST-c-Jun (1-169), hsp27 peptide ( R R L N R Q L S V A ) , human recombinant hsp27, S6-10 peptide ( R R L S S L R A ) , and casein phosphotransferase activities were measured in column fractions. For M B P , S6-10, casein and hsp27-peptide phosphotransferase activities, 5 ul of column fractions were added to 25 ul of Reaction buffer containing 20 ug of a substrate. The reactions were started by adding 50 u M [y- 3 2 P]ATP (2000 cpm/pmol). After 10 min of incubation at 30 °C, the reactions were terminated by spotting a 15-ul aliquot of the reaction mixture onto a p81 phosphocellulose filter paper (1.5 x 1.5 cm). After washing the papers 10 times with 1% phosphoric acid, they were counted in a scintillation counter in the presence of 0.5 ml scintillation fluid. The phosphotransferase activities toward human recombinant hsp27 and GST-c-Jun (1-169) were measured after column fractionation by addition of 30 ul of the fractions to 1 ug of human recombinant hsp27 or 4 ug of GST-c-Jun in Reaction buffer. Assays were started by addition of 50 u M [y- 3 2 P]ATP (2000 cpm/pmol) and incubated for 10 min at 30 °C. After termination of the reactions by addition of 5 x Sample loading buffer, the hsp27 and GST-c-Jun proteins were resolved in 11% S D S - P A G E gel and the radioactivity present was quantified as described in Section 4.3.11.1. 4.3.11.3. Immunoprecipitation and determination of protein kinase activities. Various protein kinases were immunoprecipitated from 300 jig of crude extracts by incubating in 3% N E T F (100 m M NaCl, 5 m M E D T A , 50 m M Tris-HCl, pH 7.2, 50 m M NaF and 3% Nonidet P-40), 20 ul of protein A-Sepharose (previously blocked with 0.1% B S A and equilibrated with 3% v/v NETF) and 1-10 ug of antibodies for 3 h with constant rotation at 4 °C. The protein A-Sepharose beads were pelleted by centrifugation and washed once with 6% N E T F (containing 6% v/v Nonidet 63 p-40), twice with 0% N E T F and washed with KII buffer. The beads were then added to 30 pi of Reaction buffer containing a substrate and the reaction was started with the addition of 50 p M [y-3 2 P ] A T P (2000 cpm/pmol). After incubating the reaction mixtures at 30 °C for 15 min, the reaction was terminated by addition of 15 p i of 5 x Sample loading buffer and substrate proteins were resolved in 11% S D S - P A G E gels. Quantification of the radioactivity was performed as described in Section 4.3.11.1. 4.3.11.4. In situ MBP kinase assay: " in-gel" kinase assay. M B P phosphotransferase activity was characterized by "in-gel" kinase assays. The ventricular extracts were resolved in 11% S D S - P A G E gels that contained 0.5 mg/ml of M B P as described in Section 4.3.6. The gels were washed twice for 30 min with 20% (v/v) 2-propanol in 50 m M Tris-HCl (pH 8.0) and incubated for 60 min in 1 m M D T T and 50 m M Tris-HCl (pH 8.0). Proteins were denatured by incubating twice for 30 min in 6 M guanidine-HCl, 20 m M dithiothreitol, 2 m M E D T A , and 50 m M Tris-HCl (pH 8.0) and then renatured by incubating for 5 h three times in 50 m M Tris-H C l (pH 8.0), 2 m M E D T A , 1 m M DTT, and 0.04% Tween-20. Before performing kinase assays, the gels were preincubated in Buffer B (40 m M H E P E S - N a O H (pH 8.0), 1 m M DTT, 0.1 m M E G T A , and 20 m M MgCb). Kinase assays were started by incubating the gels for 60 min at room temperature in Buffer B containing 50 p M [y- 3 2 P]ATP (20,000 cpm/pmol) and were stopped by washing the gels with 5% trichloroacetic acid containing 1% sodium pyrophosphate. After the gel was dried, [32P]-labelled proteins were detected by autoradiography. 4.3.11.5. In vitro protein phosphorylation and autophosphorylation of the histidine kinase. Cell extracts or the partially purified histidine kinase were incubated in Buffer C containing 10 64 m M M g C h and 25 m M Tris-HCl, p H 7.5. The reactions were started by addition of 50 u M [y- P] A T P (2000 cpm/pmol) and incubated at 30 °C for 15 min. The reactions were terminated by addition of 5 x Sample loading buffer, followed by 11% S D S - P A G E without boiling samples, transblotting to nitrocellulose or P V D F membrane and autoradiography. The enzyme activities were either counted by excising bands from the membrane or by analyzing densities of the phosphoimages. 4.3.11.6. Photoaffinity labeling of PKX with azido-ATP. U V light activated photolabelling of A T P binding protein was performed by incubating partially purified extracts with 10 m M M g C k and 100 u M [y- 3 2P]azido-ATP (20,000 cpm/mol) at 0 °C. Irradiation with U V light (254 nm) at a path length of 2 cm was used to start the reaction and the reaction was terminated by adding 5 x Sample loading buffer after 2 min. The sample was subjected to S D S - P A G E and visualized by autoradiography. 4.3.12. Phosphoamino acid analysis. 4.3.12.1. Basic phosphoamino acid analysis using thin layer chromatography. 32 For identification of phosphorylated amino acids, P-phosphorylated proteins were resolved in S D S - P A G E and transferred to an Immobilon membrane. Once the 3 2P-protein was identified by autoradiography, the band was excised, cut into 1 mm strips and incubated at 105 °C in 500 ul of 3 N K O H for 5 h. The alkali-labile phosphoamino acids, notably phosphoserine and phosphothreonine, were destroyed by this step. Next, the solutions were neutralized by addition of 300 ul of perchloric acid. The neutralized hydrolysates were mixed with standard phosphotyrosine and 4 p l of the samples were applied onto a silica gel thin layer chromatography (TLC) plate. For phosphorylated histidine, arginine and lysine, T L C was performed with two successive chromatographies in solvent containing 65 ethanol (25%) and ammonia solution (3.5:1.6, v/v). The standard amino acids and P rabelled amino acids were visualized by ninhydrin and autoradiography, respectively. 4.3.12.2. Basic phosphoamino acid analysis using Mono Q column. The phosphorylated proteins were identified and hydrolyzed as described in the previous section. The hydrolyzed samples were applied on to a Mono Q column and the column was equilibrated for 2 min with 5% of buffer containing 1 M KHCO3 at pH 8.5 with the flow rate of 1 ml/min. The phosphoamino acids were eluted at the same flow rate with a linear salt gradient from 5% to 50% in 15 min and from 50% to 100% in 5 min. The fractions were collected for 1 ml/min for each vial and counted for radioactivity. The standard phosphohistidine sample (400 ul) was mixed with 4 pi of A M P and subjected through the F P L C with the same procedure as above. 4.3.13. Densitometry and statistical analysis. For the quantitative analysis of protein expression, the films obtained from the E C L detection system or the color developed membranes were scanned and the intensities of the bands were quantified by the NTH image program. Data are presented with standard error of means (± SEM) , and paired Student t-test (P<0.05), analysis of variance (two-way A N O V A ) and Tukey's test were performed with an a < 0.05 level of significance. 4.4. Purification of 40-kDa histidine kinase from bovine heart ventricle. 4.4.1. Homogenization of bovine heart ventricle. Bovine hearts were obtain from Olympia Fine Meats and transported to our laboratory on ice. Upon arrival in the laboratory, the ventricles were sliced into 1-cm thick pieces approximately with a 66 scalpel and ground with a mixer in ice-cold homogenization buffer until uniformly minced. The tissues were then homogenized on ice with a Polytron for 3 x 1 min duration. The resulting homogenates were centrifuged at 17,000 rpm (23,000 g) for 30 min to pellet particles or tissue debris. The supernatant was collected and stored at -70 °C until use. 4.4.2. Ammonium sulfate precipitation of crude extracts (see Appendix 1.1). Differential ammonium sulfate precipitation was performed on ice as described by England and Seifter (1990). Ammonium sulfate was slowly added in increments with constant stirring on ice. When all of the salt had been added, the mixture was stirred for another 15 to 30 min to allow equilibration of the solvent and protein. The mixture was then centrifuged at about 3000 g for 30 min in a pre-cooled centrifuge at 4 °C. The supernatant was decanted and the precipitates were re-dissolved with 100 ml of Mono Q buffer and stored at -70 °C until use. 4.4.3. Desalting by G25 Sephadex filtration. The 60-80% cut of ammonium sulfate precipitates (50 ml) was passed through a 180-ml G25 Sephadex (Pharmacia) column, which was equilibrated with 300 ml of KII buffer over night. Fractions were collected and the elution of proteins was monitored by Bradford protein assay. 4.4.4. Negative purification by Q-Sepharose and DEAE-cellulose column chromatography (see Appendix 1.3 & 5). The D E A E or Q-Sepharose columns (50 ml bed volume) were connected to a F P L C system. The column was then equilibrated at 10 ml/min with 250 ml of Mono Q buffer. Before loading into columns, samples were diluted with 5 x volume of Mono Q buffer and applied with a 50-ml super-67 loop. While loading the sample at 5 ml/min, 25 x 5 ml fractions were collected and the column was washed with 100 ml of Mono Q buffer containing 0.8 M NaCl. The flow-through fractions and 0.8 M NaCl eluants were pooled and assayed for kinases. Before loading on to the next column, the pooled fractions were concentrated by using a Millipore centrifugal filter device (Biomax-IOK). 4.4.5. SP-Sepharose column chromatography (see Appendix 1.4). The SP-sepharose column was packed to make 90 ml column volume and connected to F P L C . The column was equilibrated at 10 ml/min with 250 ml of KII buffer (pH 6.8). After the sample was diluted with a 5-fold volume of KII buffer, 50 ml of the diluted sample were loaded onto the column and proteins were eluted using a 200 ml linear gradient of 0.8 M NaCl in 5-ml fractions. 68 5. RESULTS 5.1. SECOND MESSENGER- AND CYCLIN-DEPENDENT PROTEIN KINASES. 5.1.1. Expression of cyclic-nucleotide dependent protein kinases: cAPK and cGPK. The cAMP-dependent (cAPK) and cGMP-dependent (cGPK) protein kinases have been implicated in the regulation of various cardiac functions. c A P K is a tetrameric holoenzyme that is activated when four c A M P molecules bind to the regulatory-subunits (R-subunits), resulting in the dissociation of catalytic subunits (C-subunits) and relief from pseudosubstrate inhibition by the R-subunits (Taylor et al, 1990; Beebe and Corbin, 1986). R-subunits are dimers, and mainly occur in two forms (type I and II). C-subunits are monomers with molecular masses of 40- to 46-kDa and occur in four isoforms, C a , Cpi, Cp2, and Cy. The m R N A of the three isoforms, C a , Cpi, and Cp2, have been detected in heart based on Northern blot analysis and on c D N A analysis using P C R (Wiemann et al, 1991; Showers and Maurer, 1986), but only the C a isoform has been detected at the protein level (Shoji et al. 1983). I immunologically investigated the presence of the C a isoform in developing heart. Affinity purified polyclonal antibody raised against N-terminus (NT) of C a subunit was used to probe ventricular extracts and the immunoreactivity was quantified by densitometry. As shown in Figure 5 A and 5B, a 42-kDa protein was readily detected in newborn heart and was down-regulated by 21% by 50 days of age. c A P K was detected in all of the tissues tested, but its level of expression varied among tissues (Fig. 5C). High amounts of c A P K were found in liver, kidney and heart (Fig. 5C). The equivalent level of expression to the heart was also detected mainly in the cytosolic fraction of isolated adult ventricular myocytes (Fig. 6). Cyclic GMP-dependent protein kinase (cGPK) is a ubiquitous homodimeric protein with 69 Figure 5. Expression of cyclic-nucleotide dependent protein kinases during postnatal development of rat ventricle. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with antibodies specific for c A P K (A) and c G P K (D, E) . The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (B, F). Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level or above, the means of any two groups underscored by the same line are not significantly different. For tissue comparison, extracts (50 ug of total protein) of 11 adult rat tissues (adipocytes (Ad), brain (Br), heart (Ht), intestine (In), kidney (Kd), liver (Lv), lung (Lu), skeletal muscle (Skm), spleen (Sp), testis (Ts), and thymus (Th)) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with c A P K - N T (C) and c G P K - C T (G) antibodies. i 70 cAMP-dependent Protein Kinase 46 kDa -cAPK - , -42 kDa -I 1 10 20 50 Age (days) 365 cAPK 42 kDa J anti-cAPK-NT Ad Br Ht In Kd Lv Lu Skm SpTsTh cGMP-dependent protein kinase cGPKG 70 kDa X cGPKG 78 kDa X Relative intensity 1.5-Relative intensity 1 -0.5 -1 anti-cGPKIa-NT F 1 .10*20«50«365 PKG-CT I PKG1a-NT mm 1 10 20 50 Age (days) 365 cGPK 78 kDa 78 kDa G ¥ mm- -X H ^ — _ "^ r Ad Br Ht In Kd Lv Lu Skm SpTsTh anti-cGPK-CT anti-cGPK-ip CT 71 1 day old Isolated adult ventricle cardiomyocytes Cyt. Part. Cyt. Part. cAPK-Isolated adult cardiomyocytes Cyt. Part. CDK8 cGPK-Kkialre CaMPK CDK7 PKC-a PKC-i anti PKC-a Wee1 anti Wee1 -X Figure 6. Subcellular expression of second messenger- and cyclin dependent-protein kinases in 1-day old ventricles and isolated ventricular myocytes. Cytosolic (Cyt.) and particulate (Part.) extracts (100 ug protein) from 1-day ventricular and isolated adult (50-day old) venticular myocytes were subjected to 11% S D S - P A G E and Western blot-ted for immunoreactivity against antibodies for c A P K , c G P K , C a M P K II, P K C - a , P K C -i , C D K 8 , K K , C D K 7 , C D K 8 and W e e l . 72 a molecular mass of approximately 78 to 80 kDa, and occurs as l a , 1(3 and II isoforms (Hofmann et al, 1992). The presence of this kinase in the heart was investigated previously and the major isoform was suggested to be l a (Lincoln and Keely, 1981; Mery et al, 1991; Keilbach et al, 1992). Protein expression of c G P K during the development of the heart has also been previously studied (Sandberg et al, 1989). The present study using antibodies raised against carboxy-terminus (CT) of c G P K - I a showed strong immunoreaction with two distinct bands at 78 kDa and 70 kDa (Fig. 5D). When Western blotting was performed using antibody raised against N T of c G P K - I a , only the upper band was detected (Fig. 5E). Therefore, it appeared that the 78-kDa protein was indeed c G P K - I a . The expression of c G P K - I a did not change significantly during development (Fig. 5F), which agrees with the study by Sandberg et al. (1989). The identity of the 70-kDa protein was not clear, but it may correspond to another isoform of c G P K which is present in non-cardiac myocytes, since it was not detected in the cytosolic fraction of isolated cardiomyocytes (Fig. 6). When different rat tissues were immunoblotted with the c G P K - C T antibody, both of the 70- and 78-kDa proteins were detected in heart and lung (Fig. 5G). Only the 78-kDa protein was detectable in brain, whereas the 70-kDa species was readily detectable in kidney and thymus (Fig. 5G). The presence of c G P K I-(3 and II was examined with specific antibodies in the heart, but these isoforms could not be detected (data not shown). Figure 5 H shows that c G P K I-(3 was readily detectable in intestine and less in liver and lung but not in heart. 73 5.1.2. Expression of Ca /lipid- and calmodulin-dependent protein kinases: PKC and CaMPKII. Several authors (Steinberg et al, 1995; Disatnik et al, 1994; Puceat et al, 1994; Bogoyevitch et al, 1996c; Rybin and Steinberg, 1994) have extensively investigated the expression of protein kinase C isoforms during development of the heart. These studies indicated the presence of P K C - 8 , P K C - e and PKC-£ isoforms and the level of expression of these isoforms decreased with age. The findings of the expression of P K C - a have been inconsistent, and appear to depend on which antibodies were used for the investigations. Recently, a study using four different antibodies clearly showed the expression of P K C - a in adult ventricular myocytes (Rybin and Steinberg, 1997). They also reported that a P K C antibody from Upstate Biotechnology was the most sensitive antibody without non-specific cross-reactions. I used the same antibody to investigate the expression of P K C - a during postnatal development of heart. Only an 80-kDa protein immunoreacted with the antibody (Fig. 7A) , and the intensity of this signal declined steadily to 23% in 365 days (Fig. 7C). Since activated P K C was also reported to be associated with membrane, cytoskeleton and nucleus, I further fractionated the ventricular extracts into cytosolic and detergent-soluble particulate fractions. At day 1, the amount of P K C -a in particulate and cytosolic fractions was equivalent, whereas in adults the P K C - a was exclusively localized in the cytosolic fraction (Fig. 6). Among various adult rat tissues, P K C - a was expressed in the highest level in the brain (Fig. 7E). In addition to P K C - a , I also investigated the expression of a Ca2 +-independent atypical isoform, P K C - i , which has not yet been described in the adult heart. P K C - i shares about 72% overall homology to PKC-C, (Selbie et al, 1993), and is involved in activation of the ultraviolet B-induced activated protein-1 (Huang et al 1996). The presence of P K C - i has been detected on 74 Figure 7. Expression of PKC and CaMPK-2 in rat ventricle during postnatal development. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with monoclonal antibodies specific for P K C - a (A) and P K C - i (B), and with affinity purified polyclonal antibodies raised against C a M P K - 2 N T (F) and C a M P K - 2 P C T (G). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (B, F). Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level or above, the means of any two groups underscored by the same line are not significantly different. In panel E and J, extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with P K C - a (E) and C a M P K - 2 (J) polyclonal antibodies. 75 Protein kinase C-a & -i 95 k D a -PKC-a -67 k D a -95 k D a -PKC-i -67 k D a -B anti-PKC-a anti-PKC-i 10 20 50 Age (days) PKC-a -I mm anti-PKC-a Ad Br Ht In Kd Lv Lu Skm SpTsTh 76 Calmodulin-dependent Protein Kinase-2 67 kDa — CaMPK2 -50 k D a -67 kDa -CaMPK2 -50 kDa -m 1 5 H c o ° o i i io-5 -0 G anti-CaMPK2-NT anti-CaMPK2-PCT H B CaMPK-NT HCaMPK-PCT 1 -10-20*50*365 15 10" H CaMPK-NT He 1•10*20«50«365 5 10 20 50 Age (days) 365 CaMPK2 -anti-CaMPK2-PCT Ad Br Ht In Kd Lv Lu Skm SpTsTh 77 Western blots of cultured chick embryonic cardiomyocytes, but immunohistochemical analysis indicated that it originated from co-cultured fibroblasts (Wu et al., 1996). I also investigated its expression in ventricular tissues during development and in freshly isolated adult ventricular cardiomyocytes. Immunoblotting with monoclonal antibody specific for P K C - i permitted detection of a 70-kDa protein that declined in intensity to 23% in adult ventricle (Fig. 7B, D). P K C - i was localized in both cytosolic and particulated fractions in 1-day hearts but exclusively localized in cytosolic fraction at 50 days after birth (Fig. 6). Due to the heterogeneity in the cell population of ventricular tissues, the immunoreaction with P K C - i antibody may have resulted from non-cardiomyocytes. It is, however, not clear why P K C - i was detected in the adult isolated cardiomyocytes. Possible explanations for this discrepancy include contamination of the isolated cardiomyocyte sample with non-cardiomyocytes or differences between the two species. The multifunctional Ca27calmodulin-dependent protein kinase II (CaMPKII) represents a family of protein kinases activated by Ca 2 +/calmodulin. It forms complexes with 8 to 12 subunits, which are composed of at least five homologous isozymes (a, (3, |3\ y, 8) (Braun and Schulman, 1995). Among the five isozymes, the 8 isozyme is predominantly expressed in heart and involved in beat to beat regulation of contractility of cardiac myocytes (Witcher et al, 1992). Subtypes of the isoforms (8A, 8B and 8c) have been further cloned (Schworer et al., 1993; Edman and Schulman, 1994). The three isoforms with apparent molecular masses of 58, 56 and 54 kDa on S D S - P A G E gels have been resolved by Mono Q anion exchange column chromatography of cytosolic extraction from isolated cardiomyocytes and each of these co-eluted with calmodulin-dependent kinase activity (data not shown). The three proteins may represent the 8A, 8B and 8c isoforms, respectively. Immunoblotting with the C a M P K I I - N T and C a M P K I I - P C T antibodies 78 permitted detection of two proteins of 56 and 54 kDa in ventricles, and their intensities were increased 4- to 10-fold during postnatal development (Fig. 7F, G , H , I). Even though the expression of C a M P K I I was up-regulated in the heart, the amount of C a M P K I I protein was still minimal when compared to other tissues, and the molecular masses varied, indicating distinct isoforms (Fig. 7J). The highest levels of C a M P K I I protein were detected in brain, followed by lung, skeletal muscle, spleen, testis and adipose, whereas rninimal amounts were detected in heart, intestine and kidney. Both of the isoforms were also detected in isolated ventricular myocytes and predominantly resided in the cytosolic fraction (Fig. 6). 5.1.3. Expression of cyclin-dependent protein kinases (CDKs). The C D K s are a family of Ser/Thr kinases that are closely related to the gene product of cdc2 in Schizosaccharomyces pombe and cdc28 in Saccharomyces cerevisiae (Nasmyth, 1993; Hartwell et al, 1974; Norbury and Nurse, 1992). These kinases are dependent upon specific regulatory subunits called cyclins, which undergo oscillating levels of expression during the cell cycle. In mammalian cells, at least eight subtypes of C D K s ( C D K 1 to 8) and their regulating partners (cyclin A to H and cyclin X ) have been identified and implicated in the control of cell cycle progression (Nigg, 1995; Norbury and Nurse, 1992b; M c G i l l and Brooks, 1995b). Expression of C D K 1 and C D K 2 declined rapidly after birth (Fig. 8A, B , C, E , F), which was consistent with a previous study that showed their presence only in neonatal heart (Tarn et al., 1995; Kang and Koh , 1997). Here, I have used two different antibodies raised against catalytic domain X I (an t i -CDKl-XI) and C-terminus ( an t i -CDKl -CT) of C D K 1 , both 79 Figure 8. Expression of cyclin-dependent kinases during postnatal development of rat ventricle. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with polyclonal antibodies specific for C D K 1 (A, B) , C D K 2 (E), C D K 4 (H), and C D K 5 (K). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (C, F, I, L ) . Inserted numbers and underscores in the graphs show results of the Tukey multiple comparison test. A t the 0.05 significance level or above, the means of any two groups underscored by the same line are not significantly different. In panel D , G , J, and M , extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with C D K 1 - C T (D), C D K 2 - M 2 (G), C D K 4 - H 2 2 (J), and C D K 5 - C T (M) polyclonal antibodies. 80 Cyclin-dependent kinase 1 Cyclin-dependent kinase 2 CDK1 32 kDa - L CDK1 32 kDa a n t i " CDK2 _, CDK1- 32 kDa-L-CT rr £ B C- 1_«10«20« 50*365 H CDK1-XI H CDK1-CT •^B-, , r -t'AK 1.5 CDKI-§ ~ 1 ca c XI | | 0.S 1 10 20 50 365 Age (days) 1 10 20 50 365 Age (days) CDK2 32 kDa CDK1 32 kDa J -anti-CDK1-CT Ad Br Ht In Kd Lv Lu Skm SpTsTh Ad Br Ht In Kd Lv Lu Skm SpTsTh CDK4 33 kDa - L Cyclin-dependent kinase 4 Cyclin-dependent kinase 5 " anti- CDK2 CDK4- 35 kDa H22 K anti-CDK5-CT rio.5-1 § ^ 1 o5 3 0.5 H DC C 0 L 1 •10«20«50-365 CDK2 1 10 20 50 365 Age (days) IftftJ 1 10 20 50 365 Age (days) CDK4 33 kDa - L anti- CDK5 - , CDK4- 35 k D a - 1 -H22 M Ad Br Ht In Kd Lv Lu Skm SpTsTh anti-CDK5-CT Ad Br Ht In Kd Lv Lu Skm SpTsTh 81 of which interacted with the same 33 kDa protein only in day 1 ventricular extracts (Fig. 8A, B) . A 33 kDa immunoreactive protein was evident in Western blots of adult tissues including intestine, kidney, liver, lung, spleen, thymus and testis, but was absent from skeletal muscle of adult rat tissues (Fig. 8D). The expression patterns of C D K 2 , 3 and 4 were also investigated and were found to be similar to that of C D K 1 . A l l of these C D K s were most evident in 1 day old ventricles and the expression dropped to undetectable levels after 20 days (Fig. 8E, F, H , I), except C D K 3 which was not detected in any stage of the heart development (data not shown). C D K 5 showed a similar trend to C D K 1 but was still detectable even after 20 days (Fig. 8K, L ) . Among other tissues, C D K 4 and C D K 5 were abundantly expressed in brain (Fig. 8J, M ) , C D K 1 in intestine, spleen and thymus (Fig. 8D), and C D K 2 in adipose, lung, testis and thymus (Fig. 8G). The expression and regulation of C D K 8 and CDK-related kinase, Kkialre (KK) , differed from that of other C D K s . The expression of C D K 8 did not change significantly during postnatal development (Fig. 9A, B). At the same time, C D K 8 was readily detectable in most adult tissues and was especially abundant in brain, heart, kidney, liver, and skeletal muscle and thymus (Fig. 9C). C D K 8 was mainly associated with the particulate fraction of isolated ventricular myocytes (Fig. 6). The expression of K K was investigated by using two antibodies raised against the C- and N-termini of K K . The N-terminal antibodies recognized three proteins with molecular masses of 45, 43 and 42 kDa (Fig. 9E). The total protein level of these three proteins increased as much as 2-fold during postnatal development (Fig. 9F). The 42-kDa protein also reacted with the C-terminal antibody and the immunoreactivity was greatly increased with age (Fig. 9D). In the isolated 82 Figure 9. Expression of CDK8 and Kkialre during postnatal development of rat ventricle. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% SDS-P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for C D K 8 (A) and Kkialre (D, E). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (B, F). Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level or above, the means of any two groups underscored by the same line are not significantly different. In panels E and J, extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with C D K 8 (C) and Kkialre (G) polyclonal antibodies. 83 Cyclin-dependent kinase 8 CDK8 40 kDa - L 1.5 CD >> S i « co c 1 DC .5 0.5 -B 1 •10-20-50-365 CDK8 anti-CDK8-NT 1 10 20 50 365 Age (days) CDK8 40 kDa - L anti-CDK8-NT Ad Br Ht In Kd Lv Lu Skm SpTsTh Kkialre Kkialre _ ^ p ant i. C-13 Kkialre _. E I anti-42 kDa -L- WB I f I" " I Kkialre-NT CD o £ f 2 DC . £ 1 " F 10*1 •20-365-50 Kkialre • H i l l 10 20 50 365 Age (days) Kkialre -j-42 kDa Q anti Kkialre-c-13 Ad Br Ht In Kd Lv Lu Skm SpTsTh 84 ventricular myocytes, K K was also detected and was mainly localized in the cytosolic fraction (Fig. 6). K K was also abundantly expressed in tissues including brain, liver and skeletal muscle (Fig. 9G). 5.1.4 Expression of CDK regulating kinases: WEE1 and CDK7. In addition to the need for cyclins in activation of C D K s , phosphorylation and dephosphorylation at specific sites are also required (McGi l l and Brooks, 1995; Nigg, 1995). For instance, the phosphorylation of Thr-14 and Tyr-15 of C D K 1 by Wee l inactivates the C D K l / c y c l i n B complex, whereas phosphorylation of Thr-161 by CDK-activating kinase ( C A K ) is required for its activation (Fesquet, 1993). C A K is a member of the C D K family, and has been purified as a complex of 42 kDa and 37 kDa proteins in HeLa cells (Fisher and Morgan, 1994; Jahn et al, 1994). The 42 kDa catalytic subunit is 88% homologous to Xenopus oocytes p 4 0 M O 1 5 (Poon et al, 1993; Solomon et al, 1993), which was renamed as Cdk7, and the p37 protein was identified as cyclin H (Makela et al. 1994; Fisher and Morgan, 1994). I have investigated the expression of C D K 7 and Wee l during heart development. Affinity purified C D K 7 proximal C-terminus (PCT) antibody immunoreacted with a 42 kDa protein in the ventricular extracts that was slightly up-regulated (1.5-fold) in expression after 1 day of age and remained at a constant level afterward (Fig. 10A, B) . The presence of the protein was also detected in all of the tissue extracts studied in 50-day old rat. The most abundant expression was detected in liver, whereas in the heart, the expression was quite low relative to other organs (Fig. IOC). In isolated ventricular myocytes, C D K 7 was readily detected only in the cytosolic fraction (Fig. 6). 85 Figure 10. Expression of CDK7 and Weel during postnatal development of rat ventricle. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for C D K 7 (A) and Wee l (D, E) . The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (B, F). Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level or above, the means of any two groups underscored by the same line are not significantly different. In panel C and G , extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with C D K 7 and Wee l polyclonal antibodies. 86 Cyclin-dependent kinase 7 CDK7 42 kDa anti-CDK7-PCT Ad Br Ht In Kd Lv LuSkmSpTs Th Weel Weel 95 kDa Weel 95 kDa ID" X 10 20 Age (days) Weel 95 kDa X G anti-Wee1-X Br Ht Lv Lu Skm Ts Th 87 Weel negatively regulates entry into mitosis by phosphorylating C D K 1 at Tyr-15 (McGown et al, 1995). It was originally cloned as a 49 kDa human protein and a 68-75 kDa Xenopus Weel protein (Mueller et al, 1995). However, based on Western blotting in HeLa cells, the endogenous form migrated as an approximately 95-kDa protein in S D S - P A G E and the 49-kDa protein was a catalytic fragment of human Wee l (McGowan et al, 1993 and 1995; Parker et al, 1995a). In this study, two antibodies raised against the first and last 12 residues of the Schizosaccharomyces pombe Wee l (anti-Weel-T) and kinase subdomain X of the human Wee l (anti-Weel-X) immunoreacted with a 95 kDa protein with no significant changes in intensity during development of the heart (Fig. 10D, E , F). Even though Wee l was readily detectable in rat ventricle (Fig. 10G) and cytosolic fractions of isolated cardiomyocytes (Fig. 6), the level of expression among other tissues was very low (Fig. 10G). Higher amounts were evident in brain, liver and testis. 5.1.5. Developmental regulation of CDK1 activity. In addition to the expression of various C D K s , I further investigated the regulation of C D K 1 activities during postnatal development. C D K 1 was specifically immunoprecipitated using an affinity purified polyclonal antibody directed against the C-terminus (Fig. 11 A) and its phosphotransferase activity toward histone H I (HH1) was determined (Fig. 11B). C D K 1 was especially predominant in 1-day old ventricular extracts and its activity declined significantly in 10-day old ventricular extracts and was almost undetectable in 50-day old ventricular extracts (Fig. 11C). In summary, the rat heart ventricle expresses various protein kinases in an age-specific 88 CDK1 32 kDa - L HH1 phos- _ phorylat ion 1 10 20 50 Abs Ext 10 20 Age (days) 50 Figure 11. Activity of CDK1 during postnatal development of rat ventricle. Total protein extracts (500 ug) from rat ventricles were immunoprecipitated and Western blotted with C D K 1 - C T antibody (Panel A ) and subsequently assayed for phosphotransferase activity toward histone H I (HH1). Panel B shows a representative autoradiogram with H H 1 . Panel C provides the mean quantitation of the results, expressed in fold of activity of 1-day old ventricle (mean +/- S E M ) , from 4 separate experiments. The 100% C D K 1 activity was 660 cpm/10 min. To confirm the specificity of the immunoprecipitation experiments, controls were performed in the absence of the antibody (Ext) or ventricular extracts (Ab). 89 Figure 12. Expression of second messenger and cyclin-dependent protein kinases during the postnatal development of rat hearts (1-365 day time period). For abbreviations, see List of abbreviations 90 manner. These protein kinases may be involved in distinct physical and biochemical characteristics during postnatal development. The change in expression during postnatal development of the heart is summarized in Fig. 12. The expression of cyclic nucleotide-dependent protein kinases, c A P K and cGPK, were slightly decreased or did not change. In contrast, those protein kinases closely involved in cell proliferation and cell division, such as PKCs and C D K s including C D K 1 , 2, 4, and 5, were down-regulated rapidly after 10 days of age. The decrease in the expression and activity may be important to keep cardiomyocytes terminally differentiated. Other C D K s such as C D K 7 , C D K 8 and K K , however, did not change or rather were increased. This indicates that C D K 7 and 8 may be involved in housekeeping functions, such as basal transcription and D N A repairing mechanism(s), and K K may mediate terminal differentiation of cardiomyocytes. 91 5.2. MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS DURING POSTNATAL DEVELOPMENT OF THE RAT HEART. 5.2.1. Expression of MAP kinase kinase kinases (MPKKKs) involved in the regulation of Erkl and 2: Raf, Mos, and Cot. The Raf family of protein-Ser/Thr kinases was first identified as the normal cellular counterparts of v-raf in murine sarcoma virus 3611. So far, three isoforms (Rafi , Raf A , and RafB) have been identified in mammals with distinct cellular distributions and characteristics (Daum et al, 1994; Su and Karin, 1996; Boldyreff and Issinger, 1997; Hagemann et al, 1997). Raf i is a ubiquitous cytosolic protein with an apparent molecular mass of 72 to 76 kDa. Activated Raf i phosphorylates and activates Mek ( M A P kinase/Erk kinase) 1 and 2. RafA and RafB are expressed in a more tissue-specific manner and their regulation is less well characterized. The expression of Raf i was investigated by using commercial antibodies raised against the C-terminal region of human R a f i . A n immunoreactive protein was detected with apparent molecular mass of 73 kDa (Fig. 13A) and the 73-kDa protein was specifically immunoprecipitated (Fig. 24A). Its level of expression gradually declined by 50-days to about 25% of the level detected shortly after birth (Fig. 13B). Among adult rat tissues, Raf i was abundantly expressed in lung, spleen, testis and thymus, but was almost undetectable in the heart (Fig. 13C). Antibody raised against the C-terminus of RafA revealed a 67-kDa protein that did not significantly change in intensity of immunoreactivity during development (Fig. 13D, E) . The antibody also specifically immunoprecipitated the 67-kDa protein from ventricular extracts (Fig. 24A). Unlike R a f i , RafA was abundantly expressed in the heart and the level of expression was surpassed only by the liver (Fig. 13F). RafB C-terminus antibody (CT) specifically but weakly immunoreacted with a doublet of 92- kDa protein in the ventricle 92 Figure 13. Expression of MAP kinase kinase kinase (MKKKs) involved in the regulation of Erkl and Erk2 during postnatal development of rat ventricle. Protein extracts (100 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with various antibodies for Raf l (A), RafA (D), RafB (G), Tpl2 (J, K ) , and Mos (N). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (B, E , H , L , O). Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level, the means of any two groups underscored by the same line are not significantly different. For tissue comparison, extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with Raf l (C), RafA (F), RafB (I), Tpl2 (M), and Mos (P) antibodies. 93 Rafi anti- [AJ Rafi- 1 C12 95K Rafi |B [3 I 1 s | 0.5 CD x 0 6 5 • 50 • 10 '20' 1 Raf1-C12 1 10 20 50 365 Age (days) anti-Raf1-**** •P" l 95K -Rafi C 1 2 Ad Br Ht In Kd Lv LuSkmSpTs Th RafA anti-RafA-CT IfRafA N 6 7 kDa E I 365 ' 50 ' 20 » 1 0 - 1 | 0.5 CD ^ 0 RafA-CT CD 1 10 20 50 365 Age (days) anti- [ ? ] " RafA-CT r KRafA N 6 7 kDa Ad Br Ht In Kd Lv Lu Skm Sp Ts Th RafB anti-RafB-CT •RafB ^92 kDa & i U - L - 1 0 - 2 Q - 5 0 R a f R . r y r -gO.5 1 10 20 50 Age (days) anti- fTJ RafB-CT r~ <RafB v 92kDa Ad Br Ht In Kd Lv Lu Skm SpTs Th 94 Tpl-2 anti-Tpl-2-CT anti-Tpl-2-PCT | L 11 - io• 2 0 • so-365 | Tpl-2-CT 0 Tpl-2-PCT| anti-Tpl-2-PCT 1 10 20 50 Age (days) _ •65 kDa •Tpl-2 Ad Br Ht In Kd Lv Lu Skm SpTs anti-Mos-lll ]-50 kDa -Mos Ad Br Ht In Kd Lv Lu SkmSpTsTh 95 and the protein was down-regulated to the similar extent as Raf l (about 50%) during development (Fig. 13G, H). To investigate the cell specific expression of these proteins, freshly isolated adult (50-day old rat) cardiomyocytes were fractionated into cytosolic and particulate fractions. In isolated cardiomyocytes, the same level of Raf l and RafA was also detected as in the ventricular tissues (Fig. 14). RafB, however, could not be detected in the isolated cardiomyocyte extracts (data not shown). In addition to Raf, at least two other protein kinases have been shown to indirectly activate E r k l and Erk2. The Tpl2 (tumor progression locus 2)/Cot (cancer Osaka thyroid) oncogene was cloned from hamster cells transformed with D N A extracts from a human thyroid carcinoma cell line (Miyoshi et al, 1991). It encodes two protein-serine kinase isoforms with apparent molecular masses of 52 kDa and 58 kDa, which arise from an alternative initiation mechanism with different transforming activities (Ohara et al, 1995). Tpl2 is a rat homologue which was cloned from cell lines derived from Moloney murine leukemia virus induced thymoma, and it shares about 90% amino acid identity to Cot (Salmeron et al, 1996). Little is known regarding the role and regulation of these proteins, but the over-expression of Tpl2 in Cos-1 and NIH3T3 cells activated both the Erk and J N K pathways with little effect on p38 kinase, probably by direct phosphorylation of M e k l and Sek l (Salmeron et al, 1996). A study showed that Tpl2 was expressed in various tissues, but highly expressed in terminally differentiated granular cells and mainly localized in cytosolic fractions (Ohara et al, 1995). Immunoblot analysis with both C-terminal and proximal C-terrninal (PCT) antibodies for Tpl2 detected an identical 58-kDa protein with gradually increasing densities up to 4.7-fold in 50-day old rats (Fig. 13J, K , L ) . Comparative studies of various adult rat tissues showed that Tpl2 was expressed 96 Cyt. Part. Raf1-RafA-Tpl2-Mos-Mek1-Erk1-Erk2-Rskt Rsk2-Cyt. Part. anti-Raf1-CT Tak1-anti-RafA-CT Mlk3-anti-Tpl2-PCT Mekkl-anti-Mos-lll Sek1-anti-Mek1-NT JNK2-anti-Erk1-CT JNK1-anti-Rsk1-PCT anti-Rsk2-PCT anti-Tak1-M17 anti-Mlk3 anti-Mekk1-CT anti-Sek1-CT Pak1-Pak2-Mkk3 Hog-Cyt. Part. anti-JNK2- Mapkapk2-| | anti-JNK1-C17 anti-Pak1-NT anti-Pak2-NT anti-Mkk3-NT anti-Hog-NT anti-Mapkapk2-NT Figure 14. Subcellular expression of protein kinases involved in M A P kinase pathways in isolated ventricular myocytes. Cytosolic (Cyt.) and particulate extracts (Part.) (100 ug protein) from isolated adult (50-day old) venticular myocytes were subjected to 11% S D S - P A G E and Western blotted for immunoreactivity against antibodies as indicated in the figure. 97 in most of the tissues with the highest amount found in heart, followed by kidney, liver, and skeletal muscle (Fig. 13M). Tpl2 was also expressed in cardiomyocytes and was localized in the cytosolic fractions (Fig. 14). Mos is another proto-oncogene-encoded 43-kDa cytoplasmic Ser/Thr protein kinase, which activates the E r k l and Erk2 pathway by direct phosphorylation and activation of M e k l (Pham et al, 1995). Immunoblotting with affinity purified antibody, raised against catalytic subdomain III of Mos , revealed a 43-kDa protein with a gradual increase in expression by as much as 2-fold at 50-day of age (Fig. 13N, O). The highest amount of this protein was detected in the skeletal muscle, followed by heart and then brain (Fig. 13P). This protein also exclusively resided in cytosolic fractions of isolated cardiomyocytes at levels comparable to that observed in ventricular extracts (Fig. 14). 5.2.2. Expression of MAP kinase kinase kinases (MPKKKs) involved in the regulation of JNK and p38 kinase: Pak, Mlk3 and Takl. The p21-activated kinases (Pak) are a family of Ser/Thr kinases that are activated by trimeric G protem-linked receptors as well as by receptor tyrosine kinases. The details for the mechanism of activation of Pak remain to be established (Benner et al, 1995). However, the direct binding of Pak to either small G proteins, Cdc42 and Rac 1, via about 60 amino-acid long p21-binding domain (PBD) or an SH3 (Src-homology 3) domain containing adapter protein, Nek, via the N -terminal proline-rich region of Pak, have been shown to induce Pak phosphotransferase activity which then stimulates stress-related protein kinases such as p38 and J N K (Bagrodia et al, 1995; Zhang et al, 1995; Jakobi et al, 1996; Teramoto et al, 1996). In mammalian systems, at least 98 three isoforms of Pak have been identified: a 68-kDa P a k l (Pakoc), a 62-kDa Pak2 (akKy, Pak-I), and a 65-kDa Pak3 (Pak(3). The immunoblotting revealed the presence of p68 P a k l and p62 Pak2 (Fig. 15A, B , F), but the absence of Pak3 in rat ventricles (Fig. 151). These results support the previous assignments of Pak2 as a ubiquitous protein, P a k l as a brain and muscle-specific protein and Pak3 as a brain-specific protein (Sells and Chernoff, 1997). In the heart, the expression of Pak isoforms was differentially regulated during the postnatal development of rat ventricle. Antibody, Pakloc-C-19, immunoreacted with three different proteins (72 kDa, 68 kDa, and 65 kDa) (Fig. 15A), all of which were also specifically immunoprecipitated (Fig. 15E). Another antibody raised against the N-terminus of P a k l , however, only cross-reacted with the 68-kDa protein (Fig. 15B). The identity of the other two bands is unknown. The 65- and 72-kDa bands might be phosphorylated forms of P a k l or other isoforms which immunoreacted specifically with the C-terminal antibody. Immunoblotting with Pak2 and Pak3 antibodies following immunoprecipitation with P a k l a antibody from ventricular extracts indicated that the 70- and 65-kDa proteins were neither Pak2 nor Pak3. Therefore, I analyzed the expression of a 68-kDa protein during development of heart which displayed a decrease to as little as 30% of newborn levels after 50-day of age (Fig. 15C). P a k l was expressed abundantly in brain, followed by liver (Fig. 15D). In the heart, the expression compared to other tissues was very low. In contrast, the expression of Pak2 increased up to 2.3-fold after 50-day of age (Fig. 15F, G) . Similar to previous reports (Sells and Chernoff, 1997), p62 Pak2 was widely expressed in all the tissues investigated (Fig. 15H); the expression in the heart was readily detectable, but was less than in testis, thymus, spleen, kidney, liver and lung. Antibody raised against the N-terminus of Pak3 detected the protein only in brain but not in other tissue extracts, even though it strongly immunoreacted with recombinant Pak3 (Fig. 151). 99 Figure 15. Expression of p21-activated kinases during postnatal development of rat ventricle. Protein extracts (150 pg) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for P a k l (A, B) or Pak2 (F). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (C, G) . Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level, the means of any two groups underscored by the same line are not significantly different. Panel E shows the P a k l that was immunoprecipitated with Paka-C-19 antibody. In panel D , H and I, extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with P a k l - N T (D), Pak2-NT (G) and Pak3-NT (H) antibodies. 100 p21-activated Kinase 1 Paki-67 kDa J I Pakl- . 67 k D a J 10 20 50 Age (days) 1 • 10 • 20 • 50* 365 T anti-Paka-C-19 anti-Pakl-NT 365 10 20 50 Age (days) Pak1-67 kDa m anti-Pak1-NT Ad Br Ht In Kd Lv Lu Skm Sp Ts Th LP. with Pakcc-C-19 Pakl- -r 67 kDa J IgG - T —'M <mfc <«M> « t anti-Paka-C-19 10 20 50 Ab. Ext. Age (days) 101 p21-activated Kinase 2 Pakl- -62 kDa-I l l IIII M 1 10 20 50 365 Age (days) anti-Pak2-NT 10 20 50 365 Age (days) Pak2- - r 62 kDa J anti-Pak2-NT Ad Br Ht In Kd Lv Lu Skm Sp Ts Th Pak3- -65 kDa-anti-Pak3-NT Br Ht Skm Lv Th Pak3 102 Mlk3 (mixed lineage kinase 3), also known as S P R K , is a Ser/Thr protein kinase, with an apparent molecular mass of 95 kDa. In ventricular extracts, a 95-kDa protein strongly immunoreacted with a Mlk3 specific antibody raised against its C-terminal residues (Fig. 16A), with increasing intensity up to 7-fold higher than newborn levels by 50-days of age (Fig. 16B). Among other tissues, the heart expressed Mlk3 relatively a high level but much less than that of the spleen (Fig. 16C). Mlk3 was also detected in isolated cardiomyocytes and present in both cytosolic and particulate fractions (Fig. 14). T a k l (TGF(3 activated kinase 1) is a 579 amino acid protein with a molecular mass of 64 kDa (Yamashita et al, 1995). The kinase domain of T a k l shares about 30% amino acid homology to Raf i and Mekk, indicating potential involvement in the J N K and p38 signalling cascades (Yamashita et al, 1995; Fanger et al, 1997a). Western blots with C-terminal antibody specific for T a k l detected a 64-kDa protein in the ventricular extracts and its expression was decreased up to 80% over the first 50 days of lifetime (Fig. 16D, F). The identical protein and expression pattern was also detected with another C-terminal antibody (Fig. 16E, F). This protein, however, was not immunoprecipitated by either antibody. Tissue distribution of this protein among other tissues, confirmed with two antibodies, showed the highest amount in brain, followed by lung, liver, thymus and heart (Fig. 16G, H). M e k k l is a mammalian homologue of the yeast protein kinase S T E 11 and Byr2 with an apparent molecular mass of 78- to 80-kDa in S D S - P A G E gels (Lange-Carter et al, 1993). Western blotting with an N-terminus specific M e k k l antibody detected a 75-kDa protein, which was also specifically immunoprecipitated from ventricular extracts by the M e k k l - P N T 103 Figure 16. Expression of MAP kinase kinase kinase (MKKKs) involved in the regulation of JNK and p38 during postnatal development of rat ventricle. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day old rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for Mlk3 (A), Tak-1 (D, E) , and M e k k l (I). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day old ventricle and values represent the mean ± S E M from 3 to 4 separate experiments (B, F, J). Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level, the means of any two groups underscored by the same line are not significantly different. In panel C, G , F, and K , extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% SDS-P A G E and immunoblotting analysis was performed with Mlk3-C20 (C), T a k l - C T (G), Tak-M17 (H), and M e k k l - P N T (K) antibodies. 104 Mixed Lineage Kinase 3 TGFp Activated Kinase 1 95 k D a J Mlk3 ' 20 50 365 Age (days) 95 kDa •»[ Mlk3 ' I Ad Br Ht In Kd Lv Lu Skm SpTsTh anti-MLK3-C20 anti-MLK3-C20 69 kDa-Tak1-50 kDa-69 kDa-Tak1-50 kDa--'= 1 "H I 0.5 CD 0 1 0 anti-TAK1-CT anti-TAK1-M17 |F |l-10-20-50-365 HTak1-M17 HTak1-CT Hi 10 20 Age (days) 50 365 69 kDa-, Tak1-69 kDa-Tak1 50 kDa-•m anti-TAK1-CT anti-TAK1-M17 Ad Br Ht In Kd Lv Lu SkmSpTs Th Mek Kinase 1 95 kDa-Mekkl-67 kDa-L U 1 10 20 50 365 Age (days) & 4 CO <D 3 1 2 > 3 1 = 0 PI 1 • io -20 -so «365 Mekkl III i l l 1 10 20 50 365 Age (days) anti-Mekkl-PNT M e k k l - ^ - - « > 67 kDa-Ad Br Ht In Kd Lv Lu SkmSpTs Th anti-Mekkl-PNT 105 antibody (Fig. 161). Following birth, the expression in rat heart ventricles increased as much as 3-fold by 20 days of age (Fig. 16J). The tissue expression pattern of M e k k l showed that the highest amount was present in the heart (Fig. 16K), which is similar to a previous analysis by Northern blotting (Lange-Carter et al, 1993). 5.2.3. Expression of MAP kinase kinases (MAPKKs), MAP kinases (MAPKs) and their substrate kinases. 5.2.3.1. Expression of Erkl and Erk2, and their up- and downstream kinases. E r k l and Erk2 are the most studied M A P kinases in the heart, and their activation by various mitogenic stimuli correlates with hypertrophy of cardiomyocytes. The expression of M e k l , a specific upstream kinase for E r k l and Erk2 activation, was analyzed by using two M e k l specific antibodies raised against either the N-terminus (NT) (Fig. 17A) or catalytic subdomain X I region (Fig. 17B) of M e k l . Both antibodies clearly immunoreacted with a 46-kDa protein, which displayed reduced intensities following birth, by up to 40-60% in 50- and 365-day old hearts (Fig. 17C). This result was consistent with a previous report by Lazou et al. (1994). M e k l was also present and predominantly resided in the cytosolic fraction of the isolated ventricular myocytes (Fig. 14). Relative to other rat tissues, the amount of M e k l in the heart was low, whereas it was abundant in brain, liver, lung, testis and thymus (Fig. 17D). Immunoblotting studies using E r k l C-terminus ( E r k l - C T ) antibodies detected both E r k l and Erk2 in the rat heart ventricle (Fig. 17E). Similar to a previous study (Lazou et al, 1994), the Erk2 expression was reduced by about half over the first 50 days after birth (Fig. 17E, F), whereas the level of E r k l remained unchanged. Even though E r k l and 2 were expressed in all 106 Figure 17. Expression of Erkl and Erk2 and their up- and downstream kinases. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for M e k l (A, B) , E r k l / 2 (E), R s k l (H) and Rsk2 (K, L ) . The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day old ventricles and values represent the mean ± S E M from 3 to 4 separate experiments (C, F, I, M ) . Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. A t the 0.05 significance level, the means of any two groups underscored by the same line are not significantly different. In panel D , G , J, and N , extracts (50 p,g of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with M e k l - X I , E r k l - C T (G), R s k l - C 2 1 , and Rsk2-PCT antibodies. 107 Mekl Extracellularly Regulated Kinases 1 & 2 anti-Mek1-NT anti-Mek1-XI 1.5 I 1 CD I 0.5 CE „ anti-Mek1-XI [AT •50 kDa •Mekl -50 kDa -Mekl LCJ365-so-20-1 • 10 & Mek1-NT " B M e k 1 - X I 10 20 50 365 Age (days) -50 kDa -Mekl 50 kDa-Erk1-Erk2-anti-Erk1-CT 50 kDa-Erk1-Erk2-1 10 20 50 365 Age (days) Ad Br Ht In Kd Lv Lu Skm S p T s Th Ad Br Ht In Kd Lv Lu SkmSp TsTh 95K-Rsk1-67 kDa-RSK1 anti-Rsk1-C21 1 10 20 50 365 Age (days) 1 10 20 50 365 Age (days) Rsk1-67 kDa-anti-Rsk1-C21 Ad Br Ht In Kd Lv Lu Skm Sp Ts Th RSK2 95 kDa-Rsk2-67 kda-95 kDa-Rsk2-67 kDa-anti-Rsk2-C19 10 20 Age (days) Rsk2-67 kDa-Ts Th Lv Skm Ht Br anti-Rsk2-PCT 108 tissues, the level and pattern of expression between E r k l and Erk2 varied among various adult tissues (Fig. 17G). For example, in brain, both E r k l and Erk2 were equally expressed but in other tissues including heart, E r k l was more abundant than Erk2. In isolated ventricular myocytes, E r k l and 2 were detected in both cytosolic and particulate fractions (Fig. 14). Among the many proposed substrates of E r k l and Erk2, the 90-kDa ribosomal S6 kinase (Rsk) has been studied extensively in many tissues and cells (Zhao et al, 1996). Two Rsk isoforms, R s k l and Rsk2, were differently regulated in their expression: R s k l was down regulated by 50% after 50 days of age (Fig. 17H, I), whereas no change was detected in Rsk2 expression (Fig. 17K, L , M ) . Since both R s k l and Rsk2 are similarly sized and might have cross-reacted with the alternative Rsk antibodies, the Rsk isoforms were specifically immunoprecipitated and then immunoblotted. These experiments confirmed a decrease in expression of R s k l during development with no change in Rsk2 levels. Immunoprecipitated R s k l did not react with Rsk2 antibody or vice versa (Fig. 24). R s k l was widely expressed in various tissues, and was especially abundant in brain, kidney, lung, liver, spleen and testis but much less in heart (Fig. 17J). Rsk2 was also widely expressed in different tissues and the amount in heart was relatively high (Fig. 17N). R s k l and Rsk2 were also detected in the isolated ventricular myocytes and mainly resided in the cytosolic fraction (Fig. 14). The expression of Rsk3 was unable to be determined, because the Rsk3 antibody, which is only available commercially, was found to cross-react with R s k l (data not shown). 5.2.3.2. Expression of JNKs and direct upstream kinases. The stress-activated protein kinases ( S A P K , also known as c-Jun N-terminal kinase or JNK) 109 -are distinctly regulated by the S A P K specific protein kinase, S e k l . Immunoblotting with two antibodies, raised against Sek l catalytic domain X I (Fig. 18A) and C-terminus (Fig. 18B), permitted detection of a 42 kDa-protein that increased in expression up to 4-fold during development (Fig. 18C). Among 10 different tissues, the heart expressed Sek l most abundantly (Fig. 18D). In isolated ventricular myocytes, strong immunoreaction was detected exclusively in the cytosolic fraction (Fig. 14). To date, at least three isoforms of S A P kinases have been identified: S A P K a , SAPK(3 and S A P K y . S A P K a and SAPK(3 isoforms (also referred to as JNK2) both exhibit an apparent molecular mass of 55 kDa, whereas S A P K y (JNK1) has a molecular mass of 46 kDa (Cano and Mahadevan, 1995; Pelech and Charest, 1995). Based on the "in-gel" assays, both of 55-kDa and 46-kDa J N K s are activated by several stimuli (Bogoyevitch et al, 1995a; Bogoyevitch et al, 1996b; Komuro et al, 1996). Western blotting with S A P K antibodies raised against recombinantly expressed whole protein or C-terminal residues detected 55-kDa (Fig. 18E) and 46-kDa proteins, respectively (Fig. 18F). Expression of these proteins did not change during development (Fig. 18G). Both of these kinases were also detected in the isolated ventricular myocytes (Fig. 14), and a relatively high amount of JNK1 (SAPKy) was detected in heart compared to other adult tissues such as adipose, brain, skeletal muscle and testis (Fig. 18H). no Figure 18. Expression of SAPK and the direct upstream kinase Sekl. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for Sek l (A, B) , S A P K (E, F). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day old ventricles and values represent the mean ± S E M from 3 to 4 separate experiments (C, G) . Inserted numbers and underscores in the graphs show results of the Tukey multiple comparisons test. At the 0.05 significance level, the means of any two groups underscored by the same line are not significantly different. In panel D and H , extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with S e k l - C T (D) and JNK1 (H) antibodies. i l l Sekl anti-Sek1-XI -46 kDa -Sekl anti-Sek1-CT -46 kDa -Sekl & 4 CO 3 3 I 2 a 0 anti-Sek1-CT Cj 1 . io • 20«so • 365 EZ! Sekl -XI — 5 S B H B B ~ HSek1-CT 10 20 50 365 Age (days) •Sekl Ad Br Ht In Kd Lv LuSkmSpTs Th c-Jun-N Terminal Kinase 1 & 2 69 kDa-50 kDa-50 kDa-JNK2 anti-JNK1-NT CD > S 0.5 0 CD CC Gj 1 » 10 • 20 « 50 • 365 HJNK2 Pi 1 10 20 50 365 Age (days) JNK1-anti-JNK1-C17 Ad Br Ht In Kd Lv Lu Skm Sp Ts Th 112 5.2.3.3. Expression of p38 and its direct up- and downstream kinases. p38 Hog, also referred to as R K (reactivating kinase), Hog, M x i 2 , Mpk2 ( M A P kinase 2) or C S B P (cytokine-suppressive antiinflammatory drug binding protein), is another member of the M A P kinase family that is distinct from E r k l / 2 and the S A P K s . M k k 3 and M k k 6 target the phosphorylation and activation of p38 Hog (Derijard et al, 1995; Han et al., 1996; Lee et al, 1996; Moriguchi et al., 1996). No M k k 6 specific antibody was available; so only expression of M k k 3 in heart was investigated with antibody raised against the C-terminus of this kinase. M k k 3 , with the expected molecular mass of 35 kDa, was detected and was upregulated as much as 2-fold during development (Fig 19A, B) . Similarly, the expression of M k k 3 in heart was higher than in any other tissues investigated (Fig. 19C). The level measured in heart was comparable to that found in isolated myocytes, where it predominantly resided in the cytosolic fraction (Fig. 14). The expression of p38 Hog was investigated using two antibodies, raised against either the N-terminus (NT) or C-terminus (CT) of the kinase. No changes were detected in the levels of p38 Hog during development (Fig. 19D, E , F). The amount of p38 Hog was higher in heart than all other adult rat tissues examined (Fig 19G). A high level of p38 Hog was also detected in isolated ventricular myocytes, where it was mainly localized in the cytosolic fraction (Fig. 14). Mapkapk2 is phosphorylated and activated by p38 Hog. Two affinity-purified polyclonal antibodies raised against either the N-terminus (NT) or proximal C-terminus (PCT) of Mapkapk2 immunoreacted with a 60-kDa protein (Fig. 19H, I), that gradually increased up to 1.7-fold during the first 50 days after birth (Fig. 19J). In comparison with other tissues, abundant amounts of Mapkapk2 were detected in heart, but were less evident in liver and 113 Figure 19. Expression of p38 Hog and its direct up- and downstream kinases. Protein extracts (150 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and Western blot analysis was performed with affinity purified polyclonal antibodies specific for M k k 3 (A), p38 Hog (D, E) , and Mapkapk2 (H, I). The immunoreactivities were plotted against the relative density as 1 arbitrary unit for 1 day old ventricles and values represent the mean ± S E M from 3 to 4 separate experiments (B, F, J). Inserted numbers and underscores in graphs show results of the Tukey multiple comparisons test. A t the 0.05 significance level, the means of any two groups underscored by the same line are not significantly different. In panel C, G , and K , extracts (50 ug of total protein) of 11 adult rat tissues (see legend to Fig 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with M k k - C T (C), Hog-CT (G), and Mapkapk2-PCT (K) antibodies. 114 p38 Hog Ad Br Ht In Kd Lv Lu SkmSp Ts Th Ad Br Ht In Kd Lv LuSkm Sp Ts Th MAPK-activated Protein Kinase 2 67 kDa- H j Mapkapk2-51 kDa-67 kDa- J Mapkapk2- — -mm*" 51 kDa-anti-Mapk-apk2-NT anti-Mapk-apk2-PCT 1 • 1 0 - 2 0 ' 50 - 365 Mapkap2-NT m Mapkap2-PCT . 1 Mapkapk2-51 kDa-anti-Mapk-apk2-PCT Ad Br Ht In Kd Lv LJ Skm Sp Ts Th 115 skeletal muscle. The high expression of this protein is consistent with a previous report which showed high levels of Mapkapk2 m R N A in the heart and muscles (Zu et al, 1997) In summary, expression of most mitogenic protein kinases, including R a f l , RafB, M e k l , Erk2 and R s k l , were significantly down-regulated, while the stress-related signalling kinases, such as M l k 3 , M e k k l , S e k l , Mkk3 and Mapkapk2, were up-regulated during development (Fig. 20). However, most M A P kinases, including E r k l , JNKs , p38 Hog, as well as Rsk2, did not exhibit postnatal changes in expression. The proto-oncogene-encoded kinases Mos and Tpl2 were up-regulated up to 2- and 4-fold, respectively, with age. P a k l , which may be involved both in the regulation of cytoskeleton and in stress signalling, was down-regulated, but Pak2 increased only after 50 days. A l l of these proteins, except RafB, were also detected in the isolated adult cardiomyocytes at levels comparable to those found in adult ventricle. Tissue distribution studies revealed that most of the protein kinases that were up-regulated during heart development also tended to be preferentially expressed in the heart. B y contrast, the down-regulated protein kinases were generally expressed in the heart at relatively lower levels than in most of other tissues. 116 Figure 20. Expression of MAP kinase signal cascades in postnatal developing rat heart (1-365 day time period). For abbreviations, see the List of abbreviation. 117 5.2.4. MBP, GST-c-Jun and heat shock protein 27 (hsp27) phosphotransferase activities during postnatal development rat ventricle. In addition to expression of protein kinases, the activities of Erk, S A P K and Mapkapk2 in extracts of rat ventricles during development were analyzed by measuring their phosphotransferase activities toward M B P , GST-c-Jun (1-169), and hsp27, respectively. On the one hand, GST-c-Jun has been shown to be efficiently phosphorylated at Ser-63 and Ser-73 residues by J N K , but not by E r k l , Erk2 or p38 kinase. Hsp27, on the other hand, is an efficient substrate for Mapkapk2, which is preferentially activated by p38 kinase (Fresgney et al, 1994). During postnatal development, the M B P phosphorylating activity in 1 day-old rat ventricles was significantly higher than in 50 day-old rat ventricles (Fig. 21 A , B) , whereas GST-c-Jun and hsp27 phosphorylating activities were very low and did not vary significantly (Fig. 21C, D , E , F). M B P , although an efficient substrate for E r k l and Erk2, can also be phosphorylated by other protein kinases, such as Pak, Raf, and protein kinase C. Therefore, I further investigated M B P kinases by employing an "in-gel" assay. At least five protein kinases that had M B P phosphorylating activity were identified, but only the 44 kDa and 42 kDa kinases varied in phosphotransferase activity during development (Fig. 21G). Since not all kinases can be re-natured in the "in-gel" assay, M B P phosphotransferase activities were also measured after fractionating the extracts using a Resource Q column in F P L C . The Resource Q column resolved four peaks of M B P phosphotransferase activities which eluted at flow-through (I), 0.28 M (II), 0.4 M (III) and 0.52 M (VI) of a continuous N a C l gradient. Western blotting with E r k l -C T antibody indicated that E r k l and Erk2 were mainly co-eluted with peak III and partially with peak I (Fig. 22). The identity of the kinases for peaks II and V I remains to be investigated. The activity of the kinase in peak III, which may represent the activities of E r k l 118 Figure 21. Phosphotransferase activities toward MBP, GST-c-Jun, and heat shock protein 27 (hsp27) during postnatal development of rat ventricle. Extracts of 1-, 10-, 20-, and 50-day old rat ventricles were incubated with M B P , GST-c-Jun, and hsp27 in Reaction buffer, as described in Materials and Methods. Following the reaction, the substrates were resolved in 11% S D S - P A G E , electrophoretically transferred to nitrocellulose membrane and exposed to film. Panel A , C and E shows a typical autoradiography of phosphorylated M B P (A), GST-C-Jun (C), and hsp27 (E). The phosphorylated substrate bands of the membrane were excised and counted in a scintillation cocktail. The results of three separate experiments were plotted and expressed as 1 arbitrary unit for 1-day tissue extracts (± S E M ) . The average 100% values for M B P - , c-Jun- and hsp27-phosphotransferase activities were 840, 85 and 102 cpm/10 min, respectively. Panel G shows the in situ M B P phosphotransferase activity of ventricular extracts assayed as described in Materials and Methods. 119 6000 Resource Q Fraction no. Figure 22. MBP phosphotransferase activities of Resource Q fractionated extracts obtained from rat heart ventricles during postnatal development. Cytosolic ventricular extracts (2 mg protein) from 1-, 10-, 20- and 50-day old rats were applied to a Resource Q column, which was eluted with a linear 0-0.8 M N a C l gradient, and the fractions were assayed for M B P phosphotransferase activity. Data is representative of two separate experiments (1000 cpm/10 min corresponded to 8 pmol/min/ml). The Western blot with E r k l - C T antibody (inserts) demonstrates that the majority of E r k l and Erk2 were eluted at peak III. 1 2 1 and Erk2, decreased with age. Peak V I also followed the same trends as peak III, whereas peak II was significantly higher at 10 and 20 day after birth. These results clearly indicate that, during development, several M B P phosphorylating kinases underwent changes in activities and the activities of E r k l and Erk2 were shown to be decreased. Similarly, after fractionating crude extracts on a Resource Q column, activities for S A P K and Mapkapk2 were also analyzed using GST-c-Jun peptide and GST-hsp27 proteins, respectively, as substrates. The phosphotransferase activity profiles for c-Jun and hsp27 exhibited several peaks (Fig. 23). Western blot analysis identified that S A P K a/p7y all mainly eluted at fraction #26 (0.32 M NaCl) , and 50-kDa and 60-kDa Mapkapk2 were in the flow-through and fraction # 26, respectively. However, the phosphotransferase activity profiles were not quite matched with those of Western blot analysis, which made it hard to interpret these results. That no distinct activity peaks co-eluted with Western blotting indicated either low activities of these protein kinases or poor sensitivities of these assays. 5.2.5. Isoform specific activities of Raf, Erk and Rsk during the postnatal development of rat ventricle. During postnatal development of the heart ventricle, the M B P phosphotransferase activities, which may correspond to E r k l and Erk2, were down regulated (Fig. 24). Raf l and RafA (upstream kinases for E r k l and Erk2), and R s k l and Rsk2 (downstream kinases for E r k l and Erk2) were all expressed in an isoform-specific manner in ventricular myocytes (Fig. 13 and 17). To investigate the activities of these kinases, isoforms of Raf, Erk and Rsk were immunoprecipitated and their phosphotransferase activities were measured. Fig. 24A shows the 122 Figure 23. c-Jun and hsp27 phosphotransferase activities of Resource Q fractionated extracts during postnatal development of rat ventricle. Cytosolic ventricular extracts (2 mg protein) from 1-, 10-, and 50-day old rats were applied to a Resource Q column eluted with a linear 0-0.8 M NaCl gradient and the fractions were assayed for c-Jun (panel A) and hsp27 (panel B) phosphotransferase activities. The Western blot with S A P K cx/(3 and Mapkapk2-PNT antibodies (inserts) demonstrate that most of the S A P K a/p/v and the 60-kDa Mapkapk2 eluted at fraction #23. 123 Fraction no. 1 8 101418 20 2224 26 2830 38 - 55 kDa SAP kinase a/p - 46 kDa SAP kinase y Resource Q fraction no. > o ca CD CO CO J D in c: ca Q -o Fraction no. 6 10 1214 16 18 20 22 24 26 30 38 14000 -10000h JL-.6000 CD t= a r t £-£2000 U i t oL T P O 60 kDa Mapkapk2 50 kDa Mapkapk2 Resource Q fraction no. 124 Figure 24. The total and specific activities of Rafl and RafA, Erkl and Erk2, and Rskl and Rsk2 during postnatal development of rat ventricle. Total protein extracts (500 ug) of 1- and 50-day old rat ventricles were irnmunoprecipitated and immunoblotted with antibodies indicated in the figure. Panel A , D , and G shows the antibody-specific irnmunoprecipitation from rat ventricular extracts. Panel B , E and H show the fold increase in total phosphotransferase activities towards M B P (B and E) and S6-10 peptide ( R R L S S L R A ) (H) of the irnmunoprecipitated kinases. Panel C, F and I show the specific phosphotransferase activities derived from the activities divided by the amount of the protein irnmunoprecipitated. Values are the relative enzyme activities and are the mean ± S E M of 4 separate experiments. The average 100% activities for Raf l , RafA, E r k l , Erk2, R s k l and Rsk2 were 850, 700, 1200, 400, 640 and 570 cpm/10 min, respectively. 125 126 immunoprecipitation of Rafi and RafA with Rafi-C12 and RafA-C-terminus antibodies, respectively. Consistent with the previous results, the amount of Rafi immunoprecipitation was lower in 50-day old ventricles than at birth, whereas the amount of RafA did not change. Similarly, the activity of Raf i was also decreased (Fig. 24B), but no change was detected in its specific activity (Fig. 24C). E r k l and Erk2 were also specifically immunoprecipitated by E r k l - C T and Erk2-C14 antibodies, respectively (Fig. 24D). The amount of immunoprecipitated Erk2 was reduced in 50-day old ventricular extracts, while no change in the amount of E r k l was observed (Fig. 24D). The total M B P phosphotransferase activity of E r k l decreased up to 70% by 50 days after birth (Fig. 24E), which also represented a change in the specific activity during development (Fig. 24F). Both of the total and specific activities of Erk2 also declined substantially (Fig. 24E and F). R s k l showed decreases in both level of expression (as determined by immunoprecipitation, followed by immunoblotting) and activity, with no change in their specific activities, a trend similar to that observed for Raf i (Fig. 24G, H , I). 127 5.3. Regulation of M A P kinase signalling cascades in the heart. 5.3.1. Regulation of M A P kinases by cyclic-nucleotide dependent protein kinases and P K C . As discussed in Section 1.3.2, it is now well established that stimulation of P K C can lead to sequential activations of R a f i , M e k l , E r k l and Erk2 in many cell systems including cardiomyocytes. However, the role of c A P K and c G P K in the regulation of Erks and other stress-response M A P kinases, such as p38 Hog and J N K , remained to be explored. Activation of c A P K can either induce or inhibit E r k l and Erk2 activation depending upon the tissue examined, but the information for its role in the regulation of p38 and J N K is still limited. Even less data is available regarding this role of c G P K . Therefore, the present study indirectly investigated how E r k l and p38 (measured indirectly via Mapkapk2, a direct downstream target of p38) are regulated by these kinases. Isolated adult cardiomyocytes were exposed to various membrane permeable P K C , c A P K and c G P K activating molecules such as phorbol 12-myristate 13-acetate ( P M A , 1 p M for 5 min), forskolin (Fsk, 20 p M for 10 min), 8-chlorphenylthio-cAMP (8-CPT-c A M P ; 100 u M for 10 min), or 8 -Br -cGMP (100 p M for 10 min) and activities of E r k l or Mapkapk2 were analyzed from the cell extracts. Inserts in Figures 25A and 25B show specific immunoprecipitation of E r k l and Mapkapk2, respectively. The phosphotransferase activities toward M B P and hsp27 peptide ( P R L N R Q L S V A ) of the immunocomplexed enzymes were plotted, respectively. As shown in Figure 25, P M A activated E r k l about 4.7-fold in 5 min. The other stimuli had no effects on E r k l activity. However, Mapkapk2 was activated up to 2-fold by 8-Br-cGMP. These results indicate that E r k l and Mapkapk2 are regulated by separate signalling pathways, and that activation of P K C and elevation of c G M P levels can lead to the activation of E r k l and Mapkapk2, respectively. 128 Figure 25. Regulation of MAP kinases mediated by cyclic-nucleotides and PKC in isolated adult rat cardiomyocytes. Isolated cardiomyocytes from adult rats were treated without (CNT) and with 8-Br-cGMP (100 uJVI for 10 min), forskolin (20 u M for 10 min), 8 - C P T - c A M P (100 uJVI for 10 min) and P M A (1 u M for 5 min). E r k l and Mapkapk2 were specifically irnmunoprecipitated from cell extracts and the phosphotransferase activities of the immunocomplexed E r k l and Mapkapk2 were measured using M B P and hsp27 peptide as substrates, respectively. Panel A shows fold of activities of E r k l (mean ± S E M , n = 3). * P<0.1 vs control. Panel B shows fold of activities of Mapkapk2 (mean ± S E M , n = 4). # P<0.05 vs control. The average 100% control activity for E r k l and Mapkapk2 were 1483 cpm/15 min and 2265 cpm/15 min, respectively. Inserts show representative immunoblots of the irnmunoprecipitated E r k l and Mapkapk2. To confirm the specificity of the immunoprecipitation experiments, controls were performed in the absence of the antibodies (Ext.) or cell extracts (Abs). 129 130 5.3.2. Nitric oxide mediated activation of Erkl/2 and p38 MAP kinases in isolated rat cardiomyocytes. Nitric oxide (NO) acts as an intracellular mediator and can activate guanylate cyclase, which subsequently increases intracellular c G M P levels and activates cGPK. N O can also elicit effects on cGMP-independent pathways (reviewed in Balligand and Cannon, 1997). This study used sodium nitroprusside (SNP) as an exogenous N O donor and its effects on M A P kinases were analyzed. As shown in Figure 26A, SNP (100 uM) increased E r k l phosphotransferase activity after 5 min and activation was maximal after 10 min (3.5-fold). A M e k l inhibitor, PD98059 (20 uM), abolished this activation, which indicated the E r k l activation by SNP was mediated by M e k l . However, the activation was not significantly inhibited by a pretreatment of a guanylyl cyclase inhibitor, 1H-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ; 20 uM) for 30 min. This result may indicate that cGMP-independent pathways mediate the activation E r k l by SNP, which is consistent with the previous data which showed no activation of E r k l by 8-Br-cGMP (Fig. 25A). Instead of analyzing p38 Hog phosphotransferase activity in vitro, Mapkapk2, which is a direct downstream kinase for p38 Hog, was specifically irnmunoprecipitated and its phosphotransferase activity toward hsp27 peptide was analyzed. As shown in Figure 26B, SNP activated Mapkapk2 within 5 min (1.5-fold) which was returned to basal levels by 15 min. Since 8-Br-cGMP was also able to activate Mapkapk2 (Fig. 25B), the activation by SNP was likely mediated by a cGMP-dependent pathway. Indeed, the activation by SNP was inhibited by a pretreatment of O D Q (20 uM). The activation of Mapkapk2 was also inhibited by 25 u M of SB203580, a p38 Hog inhibitor, which implies the Mapkapk2 activation was also dependent on p38 Hog activation (Fig. 26B). 131 Figure 26. Cyclic-GMP dependent- and independent-activation of MAP kinases by SNP in isolated adult rat cardiomyocytes. Isolated cardiomyocytes from adult rats were treated with sodium nitroprusside (SNP, 100 p M ) . Inibitors, lH-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ; 20 pJVI), PD98059 (20 pM) and SB203580 (25 pM) were added 30 min before the treatment. E r k l and Mapkapk2 were specifically immunoprecipitated from cell extracts and the phosphotransferase activities of the immunocomplexed E r k l and Mapkapk2 were measured using M B P and hsp27 peptide as substrates, respectively. Panel A shows fold stimulation of the phosphotransferase activities of E r k l (mean ± S E M , n = 5 for SNP, n=3 for O D Q and n=2 for SNP + PD98059). * P<0.5 vs control. N S : at the 0.05 significant level, the means of the two groups are not significantly different based on the paired t-test. Panel B shows the fold stimulation of the phosphotransferase activities of Mapkapk2 (mean ± S E M , n = 6 for SNP, n=3 for O D Q and n=2 for SNP + SB203580 (range is shown instead)). # P<0.05 vs control. S: at the 0.05 significant level, the means of the two groups are significantly different based on the paired t-test. The average 100% control activity for E r k l and Mapkapk2 were 1483 cpm/15 min and 2265 cpm/15 min, respectively. 132 o ca a> cn ca "S5 cz as o C L , — . o CNT 5 min 5 min+ODQ 10 min 15 min 5 min SNP 1 1 + PD98059 + SNP CNT 5 min 5 min+ODQ 10 min 15 min L + SNP 5 min SNP + SB203580 133 5.3.3. Regulation of Erkl by endothelin-1 and adenosine in perfused whole hearts and isolated cardiomyocytes of rat heart. The activation of E r k l and Erk2 by endothelin-1 (ET1) and adenosine was investigated both in isolated adult ventricular myocytes and perfused rat heart ventricles. ET1 , a 21-amino acid peptide, was originally identified as a vasoconstrictor secreted by bovine aortic endothelial cells (Yanagisawa et al, 1988). In the heart, ET1 acts via G protein-coupled serpentine receptors and increases both contractility and gene expression (Kramer et al, 1992). In cultured neonatal cardiomyocytes, it rapidly activated E r k l and Erk2, via Raf and M e k l (Bogoyevitch et al. 1995b). Since ET1 directly targets cardiomyocytes, the regulation of E r k l and Erk2 by ET1 was studied in a suspension of isolated adult ventricular myocytes. ET1 (100 nM)-treated and control extracts of ventricular myocytes were fractionated on a Mono Q column and M B P phosphotransferase activity was measured. Mono Q column chromatography resolved a peak of M B P phosphotransferase activity at fraction # 14 (0.4 M NaCl), which was maximal in the extracts of 5 min ETl-treated myocytes (Fig. 27A). To investigate whether this activity was attributable to E r k l , the anti-Erkl C-terminus antibody was used to immunoprecipitate the protein from the peak fraction and its phosphotransferase activity of the immunocomplexed protein was measured. The results clearly showed that E r k l was immunoprecipitated from fraction # 14 and activated maximally at 5 min. The activity returned to basal level by 15 min (Fig. 27B). Similarly, the effect of adenosine on E r k l and Erk2 activities was also investigated. Adenosine, which is derived from cardiomyocytes and vascular endothelium during ischemia, exerts vasodilation and negative inotropic and chronotropic effects on vascular smooth muscle and cardiomyocytes, respectively (Mullane and Bullough, 1995). In isolated perfused rabbit heart, 134 Figure 27. Activation of Erkl by endothelin 1 in isolated adult rat ventricular myocytes. Isolated ventricular myocytes from adult rat heart were treated with 100 n M endothelin-1 for the times shown. Panel A , cytosolic extracts (1.5 to 2 mg protein) from control ( • ) , 3 min (O), 5 min (o) and 15 min ( A ) endothelin-1 treated isolated ventricular myocytes were applied to a Mono Q column that was eluted with a linear 0-0.8 M N a C l gradient in 500 u l fraction size. Fractions (5 pi), # 8 to 25, were assayed for phosphotransferase activities toward M B P . Data is representative of three separate experiments. Panel B , E r k l was irnmunoprecipitated with E r k l -C T antibody from Mono Q fractions of control (dotted), 3 min (grey), 5 min (dark grey) and 15 min (black)-treated isolated ventricular myocytes extracts and subsequently assayed for M B P phosphotransferase activities. Results show an average of the M B P phosphotransferase activities of two separate experiments (1000 cpm/10 min corresponded to 8 pmoVmin/ml). 135 136 ischemia results in the increased release of adenosine by as much as 16-fold (0.32 u M baseline to 5.12 uM) and can precondition the heart (Cohen et al, 1995). In the present study, isolated rat hearts were perfused with buffer containing adenosine (20 uM), and the ventricular tissue extracts were assayed for E r k l and Erk2 activities after fractionating the crude extracts on a Resource Q column. As shown in Fig. 28A, E r k l and Erk2 were activated by adenosine at 5 min. At the same time, E r k l and Erk2 were irnmunoprecipitated by E r k l C-terminus and Erk2 C-14 antibodies, respectively, from crude extracts of the ventricles and consistently showed activation of both kinases at 5 min (Fig. 28B). Western blotting of the immunoprecipitates showed that both E r k l and Erk2 were specifically irnmunoprecipitated in the same amounts. Since the observed activation could be due to non-cardiac myocytes, such as endothelial tissues, I further investigated whether the activation was also present in isolated cardiomyoctes. As shown in Figure 28C, adenosine indeed activated E r k l maximally at 5 min, after which the activity was diminished gradually. These results clearly indicate that adenosine can activate E r k l and Erk2 in cardiomyocytes. 5.3.4. Activation of Mapkapk2 by adenosine and other stimuli. Adenosine activated Mapkapk2, within 5 min of stimulation. Figure 29A shows maximal phosphorylation of human recombinant hsp27 following a 5 min adenosine treatment (20 uM) of perfused isolated rat hearts. When the extracts were further fractionated on a Resource Q column, peaks of adenosine-activated hsp27 kinases were eluted at two different positions in the NaCl gradients: 0.32 M and 0.48 M (Fig. 29). As previously shown, Western blots of these fractions using Mapkapk-2 P C T antibody indicated that the 50-kDa isoform was mainly eluted in the flow-through, whereas the 60-kDa isoform was detected in 0.32 M NaCl fractions (Fig 23). The 137 Figure 28. Activation of Erkl and Erk2 by adenosine in isolated perfused whole heart and isolated cardiomyocytes of rat. Isolated adult whole hearts were perfused with adenosine (20 p M ) for 1 rnin ( O ) , 5 min (0) and 15 min ( A ) , and fast frozen in liquid nitrogen. As a control heart ( • ) , hearts were perfused for 5 min without adenosine. The extracts (1.5 to 2 mg of protein) were applied to Mono Q column that was eluted with a linear 0-0.8 M N a C l gradient in 250 p i fraction size. Panel A , fractions (5 pi) were assayed for phosphotransferase activities toward M B P . Data is representative of two separate experiments. Panel B , E r k l and Erk2 were immunoprecipitated with a mixture of E r k l -C T and Erk2-C14 antibodies form the crude extracts, and subsequently assayed for M B P phosphotransferase activities. Data for control and 5 min shows the mean + S E M (n = 3). Data for 1 min and 15 min are the result of one experiment. *P<0.05 vs control. Panel C, E r k l was immunoprecipitated with a E r k l - C T antibody from the crude extracts of isolated cardiomyocytes and assayed with the same method as Panel B . Data shows the mean ± range (n = 2). To confirm the specificity of the immunoprecipitation experiments, controls were performed in the absence of the antibody (Ext) or cell extracts (Ab). 138 & 6000 I 5000-1 CD £-= 4000-1 | ° 3000 •§.2:2000 CD O m 1000 Time (min) 139 Figure 29. Activation of Mapkapk2 by adenosine in isolated perfused adult rat heart ventricle and isolated adult rat cardiomyocytes. Isolated adult whole hearts were perfused with adenosine (20 u M ) for 1 min, 5 min and 15 min, and fast frozen in liquid nitrogen. Control hearts were perfused for 5 min without adenosine. Panel A shows a representative phosphoimage of phosphorylated human recombinant hsp27 from crude extracts. Similar results were obtained in three separate experiments. The extracts (1.5 to 2 mg protein) were applied to a Mono Q column eluted with a linear 0-0.8 M N a C l gradient in 250 u l fraction size. The Mono Q fractions (5 ul) were assayed and the phosphoimages were developed as before. Panel B and C show peaks of hsp27 phosphotransferase activities at fraction #18-24 and # 34-38, which correspond to 0.32 M and 0.48 M N a C l gradients, respectively. Data is representative of two separate experiments. Mapkapk2 was irnmunoprecipitated with Mapkapk2-PCT antibody from the crude extracts of isolated adult whole hearts (D and E) or isolated cardiomyocytes (F), and subsequently assayed for hsp27-peptide ( R R L N R Q L S V A ) phosphotransferase activities. Data for control and 5 min of panel E shows the mean ± S E M (n = 3). Data for 1 min and 15 min of panel E are the result of one experiment. * indicates that the mean of the groups are significantly different based on the paired t-test at the 0.05 significant level. The average 100% activity of control Mapkapk2 in panel E is 220 cpm/min. Panel D , the irnmunoprecipitated extracts were immunoblotted with the same antibody, which shows that the same amount of Mapkapk2 was specifically irnmunoprecipitated from each extract. Panel F shows the mean ± range (n=2). The average 100% activity of control Mapkapk2 in panel F was 183 cpm/min. To confirm the specificity of the immunoprecipitation experiments, controls were performed in the absence of the antibody (Ext) or cell extracts (Ab). 140 Control Hsp27 -Hsp27 -Ventricular extracts Hsp27 - - 2 7 kDa 1 5 15 Time (min) B 4 P > <MP> <4H>> HW/t* " 5 min C 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Mono Q fraction no. AB CNT 1 15 EXT IgG Mapkapk2 <u 0 ) to in E | £ o « 8- o Q . o CM a. CO (U </> CO . a— — CO > . I£ i= O o <0 f o Q T 3 - O CNJ M ~—' CNT 5 10 15 Abs/Ext Time (min) 141 identity of the hsp27 kinase that eluted at 0.48 M NaCl is presently unclear. The ventricular extracts were also irnmunoprecipitated with affinity purified polyclonal antibody specific to Mapkapk2 and kinase activity was measured. As shown in Figure 29E, the Mapkapk2 activity towards a hsp27 protein also peaked at 5 min, which was consistent with the results shown in Figure 29A and C. Western blot analysis indicated that the 50-kDa Mapkapk2 was specifically immunoprecipitated from each crude homogenate (Fig. 29D). However, the presence of 60-kDa Mapkapk2 was not verified due to a co-migration with IgG that interacted with the secondary antibody during Western blotting. Similarly, adenosine was also used to treat isolated adult cardiomyocytes and Mapkapk2 phosphotransferase activity was measured. As shown in Figure 29F, adenosine activated Mapkapk2 in 5-10 min maximally. In addition to adenosine, anisomycin and hyperosmolarity were used to "stress" perfused rat hearts and the phosphotransferase activities toward hsp27 peptide were measured. Phosphotransferase activity assays toward hsp27 peptide from Resource Q column fractions showed a robust activation in fraction #26 (Fig. 30), which corresponded to the 60-kDa Mapkapk-2 isoform (Fig. 23). However, these stimuli had less effect on hsp27 kinase in fractions #32 and 34 (Fig. 30). 142 Figure 30. Regulation of Mapkapk2 by anisomycin and sorbitol. Isolated adult hearts were perfused with anisomycin (50 ng/ml, O) or sorbitol (0.5 M , A ) for 15 and 5 min, respectively, and fast frozen in liquid nitrogen. Control hearts (•) were perfused for 5 min without any agonist. The extracts (1.5 mg protein) were applied to a Resource Q column that was eluted with a linear 0-0.8 M N a C l gradient. Fractions (5 pi) were assayed for phosphotransferase activities toward hsp27 peptide ( P R L N R Q L S V A ) . Data is representative of two separate experiments (1000 cpm/10 min = 8 pmol/min/ml). 143 5.4. INSULIN REGULATED PROTEIN KINASES DURING POSTNATAL DEVELOPMENT OF RAT HEART. 5.4.1. In vivo insulin activation of protein-serine kinases in rat heart. Insulin targets the heart and regulates its functions via binding to and activating receptors, subsequently initiating cascades of protein kinases (Brownsey et al, 1997). In various other target cells, such as skeletal muscle, adipose tissue and liver, activation of insulin receptors induces activity of various protein kinases including PI3-K, P K B , S6K, casein kinase 2 (CK2) and M A P kinases which are involved in the regulation of metabolic enzymes, gene expression and protein synthesis. To investigate the protein-serine kinases that might be relevant to the actions of insulin in the adult rat heart, a maximal physiological dose of insulin (i.e. 2 U/Kg) was administered to overnight fasted, 50-day old rats by tail vein injection and the hearts were subsequently excised for the preparation of ventricular extracts. Previous studies in this laboratory have determined that optimal insulin activation of P K B and S 6 K in rat skeletal muscle occurs within 5 min of insulin injection. Similar results were also obtained for rat heart ventricles, where the P K B phosphotransferase activity toward M B P was elevated 4-fold by 2 min post-insulin injection (Fig. 31 A ) . The S 6 K phosphotranserase activity toward an S6 C-terminal peptide was stimulated 2-fold after 5 min of insulin treatment (Fig. 3 ID). This reflects an increase in the specific enzyme activities of these kinases, since the total amounts of P K B and S 6 K protein were unchanged in these immunoprecipitation experiments (as shown by Western blotting; Fig. 3 I B , E) . C K 2 has also been reported previously to be modestly activated following insulin treatment in several mammalian cell lines and rat tissues, although these observations have been disputed 144 Figure 31. Activation of protein kinase B (PKB) and p70 S6 kinase (S6K) by insulin in rat ventricle. Total protein extracts (500 pg) from control (saline injected) and 2 and 5 min insulin (2 U/kg)-treated adult rat ventricles were immunoprecipitated with either P K B - P H antibody (Panels A-C) or S6K-CT antibody (Panels D-F), and subsequently assayed for phosphotransferase activity toward myelin basic protein (MBP) or an S6 peptide ( R R L S S L R A ) . Panels C and E show representative autoradiograms with M B P or the S6 peptide, respectively. Panels A and D provide the mean quantitation of the results from 4 separate experiments where the autoradiograms of M B P and S6 peptide were scanned and expressed as arbitrary units (mean ± SEM) . Panels B and E show Western blots of the immunoprecipitates were probed with P K B - C T antibody and S6K-NT antibody, respectively. To confirm the specificity of the immunoprecipitation experiments, controls were performed in the absence of the antibodies (Ext) or ventricular extracts (Ab). *P<0.05 vs control. 145 PKB " IgG -MBP -0 2 5 Time post-insulin (min) 0 2 5 Ext Ab IBI " 200 2 t _o CO co -p ° - > , CD . - ^ ^ — CD ™ C L CD co <2 CD D S6K 1* 1 1 I 1 1 1 0 2 i 5 Time post-insulin (min) 0 2 5 Ab Ext S6K -IgG -S6 peptide -146 (Hei et al. 1993; Maeda et al. 1991; Issinger, 1990). To test whether insulin activated C K 2 in rat heart, ventricular extracts were subjected to anion-exchange chromatography. Phosphotransferase activity toward casein was eluted from a Resource Q column with approx. 0.5 M NaCl (i.e. fractions 19 and 20 in Fig. 32A). This activity could be attributed to C K 2 based on its sensitivity to heparin inhibition and its coelution with immunoreactivity to a CK2-specific antibody (Fig. 32A insert). Insulin evoked a modest 40% increase in the casein phosphotransferase activity of C K 2 5 min following injection into the rats (Fig. 32B). Another family of protein-serine kinases that has been commonly linked to insulin signal transduction includes Erk2 and E r k l . However, recently their roles in the metabolic actions of insulin have been challenged (Denton and Tavare, 1995). M A P kinase activation in response to insulin was evaluated both in Resource Q fractionations (Fig. 33A) and immunoprecipitated E r k l (Fig. 33B). In the fractions (#12-14), where E r k l and Erk2 eluted from the Resource Q column (Fig. 33A insert), there was no detectable increase in the M B P phosphotransferase activity as a consequence of insulin exposure for either 2 or 5 min. Measurement of the kinase activity of E r k l , after specific immunoprecipitation from unfractionated ventricular extracts of control and insulin-treated rats, also failed to show insulin activation of E r k l (Fig. 33B). These findings are consistent with a previous report that failed to detect activation of M A P kinase by insulin in cardiomyocytes (Lefebvre et al, 1996). 5.4.2. Developmental regulation of the PI3K/PKB/S6K pathway in rat hearts. Phosphatidylinositol 3-kinase (PI3K) has been implicated as an important enzyme in insulin 147 Figure 32. Activation of casein kinase 2 (CK2) by insulin in rat ventricle. Extracts (2 mg) from control ( O , * ) and 2 ( A , ^ ) and 5 min ( V , ^ ) insulin-treated adult rat ventricles were applied to a Resource Q column that was eluted with a linear 0-0.8 M N a C l gradient. Panel A , fractions were assayed for phosphotransferase activity with casein in the absence (open symbols) and presence of 10 pg/ml heparin (closed symbols). Western blotting with CK2-III antibody confirmed that C K 2 eluted in fractions 18 and 19 (Panel A insert). Panel B shows the mean results (± S E M ) from 3 separate experiments for the measurement of C K 2 . *P<0.05 vs control. 148 149 Figure 33. Lack of activation of MAP kinases Erkl and Erk2 by insulin in rat ventricle. Panel A , cytosolic ventricular extracts (2 mg protein) from control ( • ) and 2 (•) and 5 min ( • ) 2 U/kg insulin-treated 50 day old rats were applied to a Resource Q column eluted with a linear 0-0.8 M NaCl gradient in 500 ul fraction size and the fractions (5 ul) were assayed for M B P phosphotransferase activity. Data is representative of two separate experiments. Panels B and C, ventricular extracts (500 ug protein) from control, 2- and 5-min insulin-treated rats were irnmunoprecipitated with E r k l - C T antibody. Panel B shows phosphotransferase activities of E r k l toward M B P (values represent the mean ± S E M from 4 experiments). The average 100 arbitrary unit is equavalent to 95 cpm/min. The Western blot with E r k l - C T antibody in Panel C demonstrates that similar amounts of E r k l were irnmunoprecipitated from the ventricular extracts. To confirm the specificity of the immunoprecipitation experiments, controls were performed in the absence of the antibody (Ext) or ventricular extract (Ab). 150 CD C/J & E w c -tr o ca i— o E C/3 o >r DM 250 0 1000 750 500 ^ 0 min - • 011 1213 1415: fraction number 2 min - • p44 p42 ERK1-ERK2-5 min - • A 10 15 Resource Q fraction number 20 25 IgG Erkl Time post-insulin (min) 0 2 5 Ext Ab 151 signalling, as this kinase can directly associate with the insulin receptor and IRS-1. It is also involved in glucose transport through regulation of Glut-4 translocation (Tanti et al, 1996; Katagiri et al, 1997). PI3K expression in the rat heart ventricle was probed in Western blots (Fig 34A), using a monoclonal antibody raised against the p85 regulatory subunit of PI3K. Densitometric analysis revealed that expression of this subunit declined by 50% during the first year of postnatal development (Fig. 34B). However, the difference in PI3K expression between 1 day old and 50 day old hearts was not statistically significant. The adult heart was a minor organ for p85 PI3K expression, with much higher levels were detectable in adipose, brain, liver, lung, spleen and thymus (Fig. 34C). Within adult rat cardiomyocytes, PI3K was principally detected in the cytosolic fraction (Fig. 35), where the enzyme is thought to reside in an inactive form (Kapeller and Cantley, 1994). The developmental regulation of P K B in heart was examined next. Using two different affinity-purified, polyclonal rabbit antibodies directed against either the pleckstrin-homology (PH) domain or the C-terminus of P K B , an appropriately sized 60-kDa protein was visualized in immunoblots of rat ventricular extracts from 1 to 365 day old rats (Fig. 36A & B) . Although the expression of this protein was downregulated by up to 60% in one year old, as compared to newborn rats (Fig. 36C), the specific-enzyme activity of irnmunoprecipitated P K B from heart was unchanged during postnatal development (Fig. 36D). Cardiac expression of P K B was comparable to levels found in other adult rat tissues tested (Fig. 36E). Within rat cardiomyocytes, the P K B resided principally in the cytoplasmic fraction, although the active form of P K B is believed to be membrane-associated (Alessi and Cohen, 1998). 152 PI3K-67 kDa-A 1 10 20 50 365 Age (days) anti-PI3K 1 10 20 50 365 Age (days) PI3K-67 kDa-anti-PI3K Ad Br Ht In Kd Lv Lu Skm Sp Ts Th Figure 34. Expression of PI3K during postnatal development of rat ventricle. Panel A , protein extracts (100 pg) from 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with a mouse monoclonal antibody against the 85-kDa subunit of PI3K. Panel B shows the means ± S E M (n=3) of the relative immunoreactivities of the 85-kDa subunit of PI3K as deter-mined by densiometric analysis. Means that were not significantly different with an a value of more than 0.05 are underscored by the same grey bar. In Panel C, extracts (50 pg of total protein) from 11 adult rat tissues (see legend to F ig . 3) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with the P I 3 K monoclonal antibody. 153 Cyt. Part. PI3K-PKB S6W GSK3p-CK2-anti-PI3K anti-PKB-PH anti-S6K-CT anti-GSK3f3-11 anti-CK2-III Figure 35. Subcellular expression of insulin-regulated protein kinases in isolated ventricular myocytes. Cytosolic (Cyt.) and particulate extracts (Part.) (100 ug protein) from isolated adult (50-day old) ventricular myocytes were subjected to 11% S D S - P A G E and Western blotted for immunoreactivity against antibodies for PI3K, P K B , S6K, G S K 3 p and C K 2 . 154 Figure 36. Expression and activity of P K B during postnatal development of rat ventricle. Protein extracts (100 ug) of 1-, 10-, 20-, 50-, and 365-day old rat ventricles were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with P K B - P H (Panel A) and P K B - C T (Panel B) polyclonal antibodies. Panel C shows the means ± S E M (n=3) of the relative immunoreactivities with the P K B - P H and P K B - C T antibodies as determined by densiometric analysis. Means that were not significantly different with an a value of more than 0.05 are underscored by the same grey bar. In panel D , P K B was irnmunoprecipitated from the ventricular extracts prepared from 1 to 50-day old rats, and subsequently assayed for M B P phosphotransferase activity. Values are the relative enzyme activities and are the mean±SEM of 4 separate experiments. In Panel E , extracts (50 ug of total protein) from 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with the P K B - P H polyclonal antibody. 155 67 kDa-PKB-50 kDa-67 kDa-PKB-50 kDa-B anti-PKB-PH anti-PKB-CT H * 0-5 67 kDa-PKB-10 20 Age (days) anti-PKB-PH Ad Br Ht In Kd Lv Lu Skm Sp Ts Th 156 The postnatal S6K expression pattern in rat ventricular extracts was investigated using antibodies raised against either the N - or C-termini of this kinase (Fig. 37A & B ) . With both antibodies, the expression of S6K was observed to be modestly (~25%) reduced during the first year of postnatal development (Fig. 37C). There was also no significant change in the basal specific enzyme activity of immunoprecipitated S6K (Fig. 37D). Adult rat heart exhibited one of the highest levels of S6K measured, with quantities that were only exceeded by brain (Fig 37E). In cardiomyocytes, S6K was present in both the cytosolic and particulate fractions (Fig. 35). 5.4.3. Developmental regulation of GSK3 and CK2 in rat ventricles. G S K 3 has been proposed to be directly phosphorylated and inactivated by P K B and S6K. This could account, in part, for how insulin prevents phosphorylation and inactivation of glycogen synthase (Sutherland et al., 1994; Cross et al., 1995; Burgering and Coffer, 1995). The relative expression of the (3-isoform of G S K 3 was low in adult rat heart, as compared to brain, liver, lung and testes (Fig. 38C). Within the myocytes, GSK3(3 was principally found in the cytoplasmic fraction (Fig. 35). No significant changes in the level of GSK3(3 expression were detected during the first year of postnatal development in rat ventricles (Fig. 38A & B) . In contrast to G S K 3 p \ ventricular expression of C K 2 was decreased up to 63% between days 10 and 365 in the rat (Fig. 39B & C) . The reduced casein phosphotransferase activity in older rats, measured after Resource Q fractionation (Fig. 39A), could largely be attributed to a decrease in the amount of C K 2 protein. In the neonatal and adult rat heart, the 42-kDa a subunit was the principal isoform detected in Western blots (Fig. 39B). The higher molecular mass species at 44-157 Figure 37. Expression and activity of p70 S6K during postnatal development of rat ventricle. Protein extracts (100 pg) of 1-, 10-, 20-, 50-, and 365-day old rat ventricles were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with S 6 K - N T (Panel A) and S 6 K - C T (Panel B) polyclonal antibodies. Panel C shows the means ± S E M (n=4) of the relative immunoreactivities with the S 6 K - N T and S 6 K - C T antibodies as determined by densiometric analysis. Means that differed with an a value of more than 0.05 are encompassed by the same grey bar. In panel D , p70 S 6 K was immunoprecipitated from the ventricular extracts prepared from 1 to 50-day old rats, and subsequently assayed for S6-10 peptide ( R R L S S L R A ) phosphotransferase activity. Values are the relative enzyme activities and are the mean±SEM of 4 separate experiments. In Panel E , extracts (50 pg of total protein) from 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with the S 6 K - C T polyclonal antibody. 158 S6K-67 kDa-S6K-67 kDa-B _ IS6K-NTH S6K-CT 3 6 5 • 5 0 • 10 • 20 • 1 4m£ anti-S6K-NT anti-S6K-CT 10 20 50 Age (days) 365 10 20 Age (days) S6K-67 kDa-anti-S6K-CT Ad Br Ht In Kd Lv Lu Skm Sp Ts Th 159 Figure 38. Expression of GSK3 during postnatal development of rat ventricle. In Panel A , protein extracts (100 ug) of 1-, 10-, 20-, 50-, and 365-day rat ventricles were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with GSK3(3-11 polyclonal antibodies. Panel B shows the means ± S E M (n=3) of the relative immunoreactivities with G S K 3 f 3 - l l antibodies as determined by densiometric analysis. In Panel C, extracts (50 pg of total protein) from 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% SDS-P A G E and immunoblot analysis was performed with the GSK3(3-11 polyclonal antibody. 160 67 kDa-GSKI3-anti-GSK3(3-11 10 20 50 Age (days) 365 & 1 to §0.75 o 0.5 10.25 CD °= 0 m r i i 67 kDa-GSKp-10 20 50 Age (days) 365 Ad Br Ht In Kd Lv Lu Skm Sp Ts Th anti-GSK3P-11 161 Figure 39. Activity of casein kinase 2 (CK2) during postnatal development of rat ventricle. Panel A , cytosolic ventricular extracts (2 mg protein) from 1 day ( O , • ) , 10 day (O, • ) , 20 day ( • , • ) and 50 day (A, ^ ) old rats were applied to a Resource Q column eluted with a linear 0-0.8 M N a C l gradient and the 0.5 ml fractions were assayed for casein phosphotransferase activity in the absence (open symbols) and presence (closed symbols) of 10 ug/ml heparin. Data is representative of 3 separate experiments. In Panel B , protein extracts (100 ug) of 1-, 10-, 20-, 50- and 365-day rat ventricles were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with antibody raised against subdomain III regions of C K 2 . In Panel C, the immunoreactivity was plotted against the relative density for 1-day ventricles and values represent mean ± range from 2 separate experiments. In Panel D , extracts (50 ug of total protein) from 11 adult rat tissues (see legend to Fig. 5) were subjected to 11% S D S - P A G E and immunoblotting analysis was performed with CK2-III antibody. 162 CD CO C D C CO O IS CTJ O 6000 4000 2000 0 B 1 day-o + heparin* |_ 10 day-o + heparin • 20day-n + heparin" 50 d a y - A + heparin A 0 5 10 15 20 Resource Q fraction number 25 50 kDa-CK2-33 kDa-B anti-CK2-I 10 20 50 Age (days) 365 e 1 CO I 0.75 | 0.5 1 0.25 CD = 0 r r r f f l 10 20 50 Age (days) 365 50 kDa-rrji CK2 33 kDa-anti-CK2-I Ad Br Ht In Kd Lv Lu Skm Sp Ts Th 163 kDa probably corresponded to a hyperphosphorylated form of the a subunit, whereas the weaker CK2-immunoreactive band at 38-kDa was due to the presence of a smaller amount of the a ' subunit (Litchfield et al, 1990). Adult heart had some of the highest levels of C K 2 expression detected in rat tissues (Fig. 39D). Although brain, thymus and testes also featured high concentrations of C K 2 , the a ' subunit appears to predominant in these tissues. Only intestines seemed to have levels of the a subunit comparable to those seen in heart. Within myocytes, most of the C K 2 was located in the cytosolic fraction (Fig. 35). In summary, during postnatal development of rat heart, PI3K, P K B , S 6 K and C K 2 were down-regulated (40-60%) with age (Fig. 40). B y contrast, ventricular glycogen synthase kinase-3(3 (GSK3(3) protein levels were maintained during postnatal development. Compared to other adult rat tissues, such as brain and liver, the levels of PI3K, P K B , S 6 K and GSK3(3 were relatively low in the heart. Even though C K 2 protein and activity levels were both reduced by -60% in 365 day as compared to 1 day old rats, expression of C K 2 in the adult heart was as high as detected in any of the other rat tissues. 164 ^/f Change less than 1.5 fold / ^ C h a n g e l .5-2 fold Figure 40. Expression of p70 S6 kinase signal cascades in postnatal rat heart (1-365 day period). For abbreviations, see List of abbreviations. 5.5. PHOSPHOPROTEINS INVOLVED IN THE POSTNATAL DEVELOPMENT OF RAT HEART VENTRICLES. 5.4.1. Detection and characterization of endogenous phosphoproteins from extracts of ventricular muscle and ventricular myocytes during development of rat heart. As the first step in searching for novel protein kinases or substrates for protein kinases which may be involved in the development of the heart, crude extracts of rat ventricles from 1- to 50-day old rats were incubated with [y- 3 2 P]ATP and phosphoproteins were resolved by S D S - P A G E (Fig. 41A), and quantified after autoradiography. As shown in Fig. 39A, at least 8 phosphoproteins (110 kDa, 89 kDa, 80 kDa, 65 kDa, 60 kDa, 43 kDa, 40 kDa and 35 kDa) were identified with changing degrees of 3 2P-labelling during postnatal development of the heart (Fig. 41B). These phosphoproteins could be either autophosphorylating kinases, endogenous substrates phosphorylated by one or more kinases or phosphotransferases (notably ATPases of various types). To further characterize these phosphorylations, 1-day and 50-day cytosolic extracts of rat heart ventricles were incubated in reaction cocktail in the presence of various inhibitors. In the controls, the same pattern of developmental changes of phosphoproteins was resolved by S D S - P A G E . Protein kinase inhibitor (PKI) and Compound 3 had no influence on the formation of phosphoproteins, but herbimycin (15 pM) inhibited the phosphorylation of the 110-kDa, 60-kDa and 35-kDa proteins, while genistein (0.7 mM) blocked the phosphorylation of 43-kDa and 35-kDa proteins as indicated by arrows (Fig. 42A). In addition, phosphate groups of the 40-kDa protein were unstable in the presence of pyridine (16% v/v) or when boiled for 3 min in neutral (pH 7.5) or acidic (pH 2) conditions (Fig. 42B). These results imply that the 40-kDa 166 Figure 41. Identification and characterization of phosphoproteins during postnatal development of rat ventricle. Protein extracts (60 pg) of 1-, 10-, 20- and 50-day old ventricles were incubated in the presence of [y -P 3 2 ]ATP for 10 min and the phosphorylated proteins were resolved in 11% S D S - P A G E . Panel A is a representative autoradiogram of the endogenous phosphoproteins of at least 8 separate experiments. Panel B is the average densitometry analysis of phosphoimages, expressed in fold of intensity ± S E M as 1 for p35 of 1-day old ventricles. 167 p110 •> p89 p80 1 1 1 r 0 1 2 3 4 Relative phosphorylation (arbitrary units) 168 Figure 42. Effects of various inhibitors on in vitro phosphorylation and the stability of phosphoproteins in boiling. The in vitro phosphorylation of 1- and 50-day old ventricular cytosolic (panel A and B) and particulate (panel C) fractions were analyzed in the presence of genistein (0.7 m M ; -i-gen), herbimycin (15 p M ; +herb), P K I (1 p M ; +PKI), compound 3 (100 n M ; +comp3). After the reactions, the reaction mixtures were incubated in the presence of 16% pyridine for 10 min or boiled for 3 min in neutral (pH 7.2), acidic (pH 2) or basic (pH 12) conditions. Panel A to C is a representative autoradiogram of two to three separate experiments. Arrows inserted in the panel represent reduced phosphoproteins in presence of indicated inhibitors or conditions. 169 control +gen +herb +PKI +comp3 pyrid boil acid age (day)!"! 50 I 1 50 I 1 50 I 1 50 I 1 50~1 Ii 50lll 50 H i 501 170 171 phosphoprotein contained a phosphoramidate bond. In detergent-soluble particulate extracts, a 43-kDa phosphoprotein was the most evident of at least 7 phosphoproteins which were detected (Fig. 42C). Even though the 43-kDa proteins in the cytosolic and membrane fractions co-migrate in S D S - P A G E , they appear to be different in several biochemical characteristics. The membrane 43-kDa phosphoprotein was partially sensitive to genistein (0.7 mM) and was only slightly unstable in the presence of pyridine, whereas the cytosolic 43-kDa phosphoprotein was stable in the presence of pyridine and partially unstable when boiled in acid. Among the 8 phosphoproteins detected in the cytosolic fraction, 6 phosphoproteins (110 kDa, 65 kDa, 60 kDa, 43 kDa, 40 kDa and 35 kDa) were also identified in the cytosolic fraction of isolated cardiomyocytes (Fig. 43). These proteins appear to be the same proteins observed in ventricular extracts, based on their sensitivity to inhibitors, stability in pyridine and sensitivity to boiling at different p H . 5.5.2. Identification and characterization of phosphoproteins in Resource Q fractionated extracts of rat heart ventricles. To further investigate the phosphoproteins during postnatal development of the heart, extracts of 1-50 day old rat heart ventricles were fractionated by Resource Q column. The fractions were incubated with [y- 3 2 P]ATP, followed by S D S - P A G E and autoradiography. Among the 8 phosphoproteins identified from crude extracts, only the 60-kDa, 43-kDa and 40-kDa proteins were readily detected (Fig. 44). The amounts of the labeled phosphoproteins also underwent developmental changes with similar patterns as those observed from crude extracts. The 40-kDa protein eluted in fraction #3-8 and the intensity of the autoradiograph was rapidly increased after birth. The 43-kDa and 60-kDa proteins were eluted with about 0.56 and 0.64 M 172 control +gen +herb pyrid. boil in boil in boil in pH7.2 pH2 pH 12 p110 > p65 > p60 > p43 > p40 > p35 > Figure 43. Identification and characterization of phosphoproteins in isolated ven-tricular myocytes. In vitro formation of phosphoproteins was analyzed in isolated adult ventricular cyto-solic extracts (60 ug) in the presence or absence of geninstein (0.7 m M , -i-gen) and her-bimycin (15 u M , +herb). After the reactions were stopped by adding 5 X S D S - P A G E sample buffer, the reaction mixtures were either incubated in 16% pyridine for 10 min (pyrid) or boiled 3 min in neutral (pH7.2), acidic (pH 2) or basic (pH 12) conditions. Arrows indicate a phosphoprotein sensitive to boiling and pyridine. The figure is a rep-resentative autoradiogram of two separate experiments. 173 Figure 44. In vitro phosphorylation of endogenous proteins in Resource Q fractionated extracts during development of rat ventricle. Protein extracts (2 mg) of 1-, 10-, 20-and 50-day old rat ventricular extracts were applied to a Resource Q column eluted with a linear 0-0.8 M N a C l gradient in 500 p i fraction size and the fractions (30 pi) assayed for endogenous in vitro phosphorylation. Phosphoproteins were subjected to 11% S D S - P A G E , followed by autoradiography. The panel is a representative autoradiogram of two to four separate experiments of similar results. Arrows indicate distinct 3 2P-labeled proteins. 174 175 N a C l concentrations (fraction #15 and #16-17), respectively. Consistent to the previous data (Fig. 4 IB) , the intensity of 60-kDa protein was decreased with age, whereas the intensity of the 43-kDa protein was high in 10- and 50-day old rat heart ventricles. 5.5.3. Biochemical characterization and tissue distribution of the 40-kDa protein in rat. 5.5.3.1. Preliminary biochemical characterization. The biochemical characteristics of the 40-kDa protein were investigated with the pooled Resource Q fractions (# 8-14). The phosphate bonds of the protein appeared to be unstable when boiled in neutral (pH 7.5) or acidic (pH 1) conditions (Fig. 45A) and in the presence of pyridine (Fig. 45C), which is identical to the 40-kDa phosphoprotein observed in curde extracts (Fig. 42A). The phosphate group was also stable in boiling at pH 12 (Fig. 45A). To eliminate possible non-specific binding of free phosphate, the in vitro labeling assay was performed after denaturing the protein by pre-boiling or in 2% SDS. Denaturation of the protein abolished the in vitro phosphorylation (Fig. 45A). E D T A (10 mM) inhibited the phosphorylation but E G T A (10 mM) had no effect, which may indicate that the kinase requires divalent cations but not C a 2 + (Fig. 45A). The kinase was stable when stored at -20 °C or below, whereas storing for 16 h at room temperature or at +4 °C abolished the activity (Fig. 45B). The phosphorylation was also partially inhibited by genistein (2 mM), a tyrosine kinase inhibitor, but not by herbimycin (15 pJVI) (Fig. 45C). The effects of genistein on the 40-kDa protein phosphorylation was further characterized after its purification by sequential Resource Q and Resource S column chromatography. To ensure that the ethanol vehicle had no effect on the kinase, the in vitro phosphorylation assay on Resource Q fractions was performed in the presence of different concentrations of ethanol. 176 I +23 +4 -20 -70 control +gen pyrid +herb Figure 45. Preliminary biochemical characterization of a 40-kDa phosphoprotein from adult rat ventricles. Panel A , B and C are results of experiments obtained from pooled Resource Q fractionations (#5-8) of adult rat ventricular extracts. Panel A , the in vitro phosphorylations were performed (n=2 to 5) in the pre-boiled (3 min) samples (lane 5), in the presence of SDS (2%, lane 6), E G T A (10 m M , lane 7) or E D T A (10 m M , lane 8). The stability of the phosphoproteins was tested by boiling for 3 min at neutral (lane 2), acidic (pH 2, lane 3) or basic (pH 12, lane 4) conditions. Panel B , Enzyme samples were stored at different temperatures as shown in the panel and the in vitro phosphorylation assays were performed (means +/- S E M , n=3). Panel C , in vitro phosphorylation assays were performed in the presence of genistein (2 m M , n=3), pyridine (18%, n=2) or herbimycin (15 p M , n=2), followed by 11% S D S - P A G E analysis. 177 0 10 20 30 40 E1handconc.(%) 1 10 Genistein cone. (mM) Figure 46. Effects of ethanol and geninstein in the partially purified 40-kDa protein histidine kinase in the adult rat ventricle. The 40-kDa protein-histidine kinase was partially purified with Resource Q and Resource S columns from the adult rat ventricle. Panel A , the curve shows the inhibition of autophosphorylation of 40-kDa kinase by ethanol. Panel B , the curve shows the inhibiton of autophosphorylation of 40-kDa kinase by genistein. The line was calculated by linear regression to the combined data sets on the logarithm of the dose. 178 Fig. 46A shows that the activity was not affected by up to 5% of ethanol, but was highly inhibited at 20%. This result indicated that the final ethanol concentration should be at or below 5% for studying the effects of genistein. The kinase activity was measured at a constant ethanol concentration (5%) and the phosphoproteins were measured after separating unincorporated A T P from the phosphorylated proteins by S D S - P A G E . After transblotting to nitrocellulose membranes, the 40-kDa phosphoprotein was visualized by autoradiography, and the band was excised and counted for its radioactivity in the presence of scintillation cocktail. Fig. 46B shows that genistein inhibited the 40-kDa phosphorylation in a dose-dependent fashion and 50% inhibition was achieved at a 2 m M concentration of genistein. To further investigate the optimal divalent concentrations, the phosphorylation from the same column fractionated extracts was carried out in different concentrations of C a 2 + , M g 2 + and M n 2 + . As shown in Fig. 47, the optimal concentration of divalent cations was between 5 to 15 m M of either M g 2 + or M n 2 + , but more than 5 m M of C a 2 + adversely affected the phosphorylation activity. The phosphorylation kinetics were asymptotic with time, which reached plateau in 50 min at 30 °C and the reaction proceeded much slower at 0 °C (Fig. 48). 5.5.3.2. Gel-filtration assays to investigate the protein complex. To investigate possible dimerization or multimerization of the 40-kDa protein, appropriate Resource Q fractions were pooled and loaded onto a Superose 12 gel-filtration column. The protein eluted at fraction #55 to 56 (Fig. 49B). The three standard markers, 67-kDa bovine serum albumin (BSA) , 43-kDa ovalbumin and 25-kDa chymotrypsin, were eluted in peaks I (fraction #52), II (fraction #54) and III (fraction #63), respectively (Fig. 49A). This result along 179 Figure 47. Effects of divalent cations in the autophosphorylation of 40-kDa histidine kinase. The 40-kDa protein histidine kinase was partially purified with Resource Q and Resource S columns from the adult rat ventricle. In vitro autophosphorylation was performed in presence of different concentration of divalent cations, followed by S D S - P A G E analysis. Results are counts of the excised phosphorylated 40-kDa kinase band. 180 Figure 48. Time course of 40-kDa kinase autophosphorylation reaction. Aliquots of a protein kinase assay reaction ([ATP] = 50 u M ) at 30 ° C (O)and 0 ° C (•) were taken at indicated times and the reaction stopped by addition of 5 x SDS loading sample buffer, followed by 11% S D S - P A G E . The 40-kDa kinase bands were excised and counted for activity. 181 32 38 43 46 49 52 55 58 61 66 Fraction no. I. BSA 67 kDa II. Ovalbumin 43 kDa III- Chymotrypsin 25kDa Figure 49. Partial purification of 40-kDa histidine protein kinase by FPLC gel filtration. The Mono Q fractions (200 ul) was injected into Superose 12 (HR10/30) column that was equilibrated with 0.25 M N a C l in KII buffer. The protein elution profile is shown in panel A . Fractions (0.25 ml) were assayed for in vitro phosphorylation. Panel B is an autoradiogram of the 40-kDa protein-histidine kinase. 182 with those outlined in the previous section may indicate that the 40-kDa protein was an autophosphorylating monomeric protein. 5.5.3.3. Confirmation that the 40-kDa protein is a protein kinase. To verify that the 40-kDa protein was a protein kinase, the protein from Resource Q fractions was incubated with [y- 3 2P]azido-ATP at 0 °C and the mixture exposed to U V light for 3 min. The 40-kDa species was radiolabeled when the incubation was performed at a temperature at which autophosphorylation was impaired. The [y- 3 2P]azido-ATP labeled protein also co-migrated with [y- 3 2 P]ATP labeled 40-kDa protein in S D S - P A G E gel (Fig. 50A). To eliminate possible co-migration of other radio-labeled proteins, this band was further separated by 2-dimensional electrophoresis using an isoelectric focusing gel as the first dimension, followed by S D S - P A G E . The [y- 3 2P]azido-ATP photo-labeled 40-kDa protein co-migrated exactly with [y- 3 2P]ATP-labeled 40-kDa protein, which indeed indicates that the 40-kDa protein was an A T P binding protein (Fig. 50B, C). The standard markers were electrophoresed in parallel with the above samples and the proteins were visualized by staining with 1% amido black. Panel E shows the colloidal silver staining of a 2-dimensional membrane, which show the high amount of contaminating proteins. The 40-kDa histidine kinase is indicated by arrows. These results demonstrated that the 40-kDa protein was likely an autophosphorylating protein with a p i value of 8.2-8.6. 5.5.3.4. Phosphoamino acid analysis of the 40-kDa protein kinase. 32 The P-labeled 40-kDa protein kinase from Resource Q, followed by Resource S chromatography, was resolved by S D S - P A G E and transferred to Nytran membrane prior to detection by an autoradiography (Fig. 51). The 40-kDa band was then excised and subjected to 183 Figure 50. Azido-ATP photolabelling of the 40-kDa histidine protein kinase. The partially purifed 40-kDa histidine kinase was labeled with 3 2 P by using either [y- 3 2P]azido-ATP or [y- 3 2 P]ATP. The U V light-activated labeling with [y- 3 2P]azido-ATP was performed at 0 °C for 2 min to minimize endogenous kinase activities. Autophosphorylation of the 40-kDa histidine protein kinase was also performed in parallel using [y- 3 2 P]-ATP at 30 °C for 10 min, which then was loaded on the S D S - P A G E gel with or without boiling. Phosphoproteins were resolved by either 1 dimensional S D S - P A G E gel (panel A) or 2-dimensional gel electrophoresis (panel B , C and D). For protein standards, 2-dimensional electrophoresis was performed in parallel. Panel A is an representative autodiagram of two separate experiments. Panel B is an autodiagram of one experiment. Panel E is a 32 colloidal silver staining of the membrane in panel C and the arrow indicates the place where the P labeled 40-kDa protein migrated. 184 40 kDa -°yl % % CO cn o cn cn o cn 96 kDa - i 69 kDa 46 kDa 38 kDa -I 27 kDa J [y- 3 2P]ATP CO 4 ^ -a. co o •a. "2. cn cn cn o 96 kDa -69 kDa -46 kDa -38 kDa -27 kDa -Azido-[Y-32P]ATP 96 kDa -69 kDa -46 kDa -38 kDa -27 kDa -CO 4^-"o_ co cn cn o o cn 96 kDa -69 kDa -46 kDa _ 38 kDa -27 kDa -2-D standard markers I. Conalbumin, 76 kDa, pl 6.0 II. Albumin, 66 kDa, pl 5.0 III. Actin, 43 kDa, pl 5.5 IV. Glyceraldehyde 3-phosphate dehydrogenase, 36 kDa, pl 8.4 V. Carbonic anhydrase, 31 kDa, pl 6.0 Colloidal silver staining 185 Figure 51. Phosphoamino analysis of the 40-kDa histidine protein kinase in rat heart ventricle. The 40-kDa histidine protein kinase of rat ventricular cytosolic extracts partially purified by a 32 Resource Q column was labelled with [y- P ] - A T P and, subsequently, resolved in 11% SDS-P A G E . Proteins were transblotted onto ®Nytran membrane and hydrolysed in basic condition as described in Methods and Materials. Phosphoamino acids were separated by either a T L C (panel A ) or a MonoQ column (panel B) . T L C was developed by using two successive solvent (ethanol : 25% ammonia solution, 3.5 : 1.6, v/v ) cycles. Panel A is a representative autodiagram of 4 separate experiments. Panel B , the hydrolysates were loaded onto MonoQ columns and the column was equilibrated for 2 min with 5% buffer containing 1 M K H C O 3 at p H 8.5. The phosphoamino acid were eluted at the same flow rate with the linear salt gradient from 5% to 50% in 15 min and from 50% to 100% in 5 min. The fractions were collected and counted for radioactivity. The standard phosphohistidine sample (400 pi) was mixed with 4 p i of A M P and separated through F P L C using the same procedure as above. Peak I and II represent free inorganic phosphate and phosphohistidine, respectively. 186 A p-histidine/ arginine/lysine p-tyrosine 3 2 p Phosphoamino acid analysis in TLC plate B_' 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Elution time (min) Phosphoamino acid analysis by MonoQ chromatography 187 alkaline hydrolysis for 5 h at 105 °C. Phosphoamino acids were separated from A T P and Pi either by thin-layer chromatography or Mono Q F P L C . A n alkaline stable phosphoamino acid(s), which can be either phospho-histidine, -arginine or -lysine, was detected (Fig. 51 A) . The same alkaline hydrolyzate was also loaded on Mono Q columns attached to F P L C (these experiments were performed in Dr. H .R . Matthews' lab, University of California at Davis). A radiolabeled phosphoamino acid was co-eluted with a phosphohistidine standard (peak II) (Fig. 5 IB) . Peak I represents free inorganic phosphate. The phospho-arginine and -lysine were eluted in the later time fractions (fraction # 23-27). These results indicate that the phosphorylation occurred on a histidine residue(s) and was probably due to autophosphorylation. 5.5.3.5. Tissue distribution of 40-kDa histidine protein kinase in adult rat tissues. The distribution of the 40-kDa protein kinase was investigated in Resource Q flow-through fractions of various adult rat tissue extracts and their relative levels of activity quantified by measuring the intensity of the autoradiograph. Among the investigated tissues, the highest level of activity was detected in heart, followed by skeletal muscle which had one-third of the heart activity (Fig. 52). In some tissues including adipose, intestine and spleen, the activity was absent. These results indicate that the 40-kDa-histidine protein kinase was expressed in a tissue-specific manner. 5.5.4. Purification of a mammalian 40-kDa histidine protein kinase from bovine heart ventricle. The 40-kDa histidine protein kinase was partially purified from bovine heart ventricles as shown in Appendix I. The bovine heart also expressed the 40-kDa kinase. However, the apparent molecular 188 mass in the S D S - P A G E gels was about 2 kDa higher than that of rat. The 40-kDa kinase was purified as much as 84-fold, after ammonium sulfate precipitation and Sephadex G25 desalting steps (see Table 2). In collaboration with Dr. R. Aebersold at the University of Washington, the partially purified 40-kDa histidine protein kinase was subjected to enzymatic proteolysis and mass spectrometry to identify the peptides. Some of the peptides were matched with two known proteins, namely including aspartate aminotransferase and succinyl-CoA ligase, whereas other peptides were derived from unknown proteins. 189 Figure 52. Tissue distribution of 40-kDa histidine protein kinase in Resource Q fractionated adult rat. Eleven tissue extracts (total protein of 2 mg) were applied to a Resource Q column eluted with a linear 0-0.8 M N a C l gradient, and the flow-through fractions pooled and assayed for in vitro phosphorylation. Phosphoproteins were resolved in 11% S D S - P A G E with or without boiling samples (panel A ) . Panel B is the densitometry analysis of the autodiagram, expressed in arbitrary intensity units. 190 Non-boiled Boiled o ° 1 2 3 4 5 6 7 8 1 1 2 3 4 5 6 7 8 I Adipose Brain Heart Intestine Kidney Liver Lung Sk muscle Spleen Testis Thymus Ad Br Ht In Kd Lv Lu Skm Sp Ts Th 191 Table 2. Purification table of 40 kDa histidine protein kinase from bovine heart ventricle. Purification step Total protein (mg) Total activity (cpm) Yield (%) Specific activity (cpm/mg/min) Purification factor Homogenate 10,000 Ammonium sulfate ppt. Sephadex G25 900 142280 100 10.5 1 Q-Sepharose 428 157850 110 25 2.3 SP-Sepharose 53 123919 87 156 15 D E A E Sepharose Resource S 9.2 122237 86 882 84 192 6. DISCUSSION The development of a heart involves a variety of subtle molecular and biochemical changes in different subcellular components. These changes are partly associated with the differential expression of signal transducing protein kinases and modulation of their activities during development. In this regard, the expression and activities of various protein kinases during postnatal development were investigated in the rat heart. In addition, the role of these protein kinases in the heart was studied by treating the whole heart or isolated cardiomyocytes with various stimuli, followed by analyzing their phosphotransferse activities. Several potential novel protein kinases or substrates involved in the development of the heart were also identified and characterized by an in vitro 3 2P-radiolabelling assay. 6.1. Expression of cyclic nucleotide-dependent protein kinases and their role in the regulation of MAP kinases. Even though the basal level of c A M P and c A M P dependent-protein kinase (cAPK) activity have been documented by several researchers, the regulation in expression of this kinase during development of the heart had not yet been extensively investigated. These studies showed that the basal level of c A M P decreased with age (Novak et al., 1996), and the specific activity of c A P K , which peaked at 7 days after birth (7 nmol/min/mg protein), also declined in adult rat heart (2.5 nmoVmin/mg protein) (Kuo, 1975; Haddox et al., 1979). Adenylyl cyclase (AC) undergoes isoform specific changes in expression during postnatal development. Among nine different isoforms of A C , type V I is the most abundant isoform in the fetus and the level of expression gradually declines, whereas type V follows an opposite pattern (Tobise et al, 1994; Espinasse et al, 1995). The physiological effects caused by these changes are unclear. The 193 expression of type V appears to be correlated with the higher total A C activity in adult heart (Espinasse et al, 1995), whereas the declining activity may correlate with the decreased expression of type V I . The declining basal cyclase activity during postnatal development may also have resulted from the lower expression of (3-adenergic receptors (Torbise et al, 1994). M y Western blot analysis indicated that the expression of c A P K also declined by 21% by 50 days after birth (Fig. 5B). These results indicate that c A P K is down-regulated in both expression and activity during postnatal development of the heart. The role of c A P K in cell proliferation and D N A synthesis is still controversial with contradicting observations in other tissues, but it appears to be involved in cardiac muscle specific gene expression and terminal cell differentiation during the early development of the heart (Abell and Monahan, 1973; Claycomb, 1976; Gupta et al, 1996a; Gupta et al, 1996b). In neonatal rat cardiomyocyte cell cultures, forskolin (10 u M for 8 min) is able to activate E r k l and Erk2, which is dependent on extracellular C a 2 + but independent of P K C activation (Yamazaki et al, 1997a & 1997b). Conversely, this study in isolated adult cardiomyocytes failed to show activation of these Erks and Mapkapk2 by forskolin (20 uJVI for 10 min) nor by 8 - C P T - c A M P (100 u M for 10 min) (Fig. 25 A ) . In fact, c A M P or c A P K inhibits the activation of M A P kinases in many other cell types by interfering with Ras-Rafl association or by reducing Raf l activity (Burgering et al, 1993; Cook and McCormick, 1993; W u et al, 1993; Haefner et al, 1994; Hordijik et al, 1994). Phosphorylation of Raf l by c A P K at Ser-43 reduces the affinity of Ra f l for Ras (Wu et al, 1993), and a phosphorylation of the small G protein, r ap lA , or SOS by c A P K inhibits the activation of Raf l by Ras (Burgering et al, 1993; Hordijik et al, 1994). In a few cell types, such as PC12, Swiss-3T3 and COS7 cells, c A P K induced the activation of Erks (Faure and Bourne, 1995; Froedin et al, 1994). Therefore, the regulation of M A P kinases by c A P K may be 194 specific for a cell type and the discrepancies observed between adult and neonatal cardiomyocytes may be due to age differences in the heart. As shown in the present study, most of the E r k l and Erk2 signalling components as well as c A P K were decreased in their expression during postnatal development (Fig. 13 and 17). Therefore, these decreases in protein expression during development may contribute to the lowering or loss of activation of Erks by c A M P in adult hearts. However, the explanation for the discrepancies awaits further investigation. c A P K can phosphorylate various proteins including cardiac troponin C, cardiac troponin I (cTNI), the cardiac L-type C a 2 + channel and phospholamban, and it has been implicated in the positive inotropic and chronotropic effects of the heart (Yabana, 1995; Robertson, 1982; A n et al, 1996). During postnatal development, the expression of troponin I (TNI) isoforms switches from cAMP-independent, slow skeletal muscle T N I to cAMP-dependent cardiac TNI . Age-specific phosphorylation of T N I and isometric tension generation is mediated by c A P K (Bartel, 1994). Therefore, despite the down-regulation of c A P K , it may still play a key role in the regulation of cardiac contractility. Cyclic GMP-dependent protein kinase (cGPK), in contrast to c A P K , has been proposed to be involved in cell proliferation and D N A synthesis in some tissues (Milks et al, 1974; Oey et al, 1974). However, in other cells, c G P K has been suggested to play inhibitory role in epidermal growth factor-stimulated proliferation (Yu et al, 1997). In the heart, the role of c G P K is not clearly understood, but a prolonged treatment (24 h) with S N P or 8 -Br -cGMP (a membrane permeable homologue of c G M P ) may involve in the induction of apoptosis in neonatal rat cardiomyocytes (Wu et al, 1997). The concentration of c G M P and the activity of c G P K were shown to be decreased at the early neonatal stage of heart development, which were reciprocally 195 related to those changes in c A M P and c A P K (Kuo, 1975). In the present study, Western blot analysis displayed no changes in the expression of c G P K during postnatal development of rat heart ventricles (Fig. 5F). A decrease in the expression of c A P K with no changes in c G P K provide additional support for the previous suggestion that c A P K is more intimately involved than c G P K in early postnatal differentiation of cardiac myocytes (Kuo, 1975; Claycomb, 1976). In addition, a membrane permeable derivative of c G M P , 8 -Br -cGMP, was able to activate Mapkapk2 via p38 Hog (Fig. 25B) without activating E r k l . This result may indicate an additional role of c G M P or c G P K in adult cardiomyocytes. As discussed in Section 6.6, the activation of p38 Hog and Mapkapk2 was shown to be involved in the stabilization of cytoskeletons and gene transcription in response to various cellular stresses. The role of c G M P and c G P K warrants further investigation. 6.2. Expression of protein kinase C and calmodulin-dependent protein kinase-2. Protein kinase C (PKC) isoforms represent a family of ubiquitous phospholipid-dependent protein Ser/Thr kinases that are implicated in the regulation of many intracellular processes. The functional role of P K C s in the heart has been investigated and reviewed by several investigators (Steinberg et al. 1995; Sugden and Bogoyevitch, 1995; Bogoyevitch and Sugden, 1996). Several neurotransmitters, growth factors, tumor promoters and mechanical stresses induce activation of P K C , resulting in an increase in gene expression and protein synthesis. One of the mitogenic pathways induced by P K C involves the activation of E r k l and Erk2 (Fig. 25A), which perhaps may contribute to the induction of hypertrophy of the heart (Bogoyevitch et al, 1995b; Gillespie-Brown et al., 1995; Sadoshima et al, 1995). 196 In the heart, C a M P K I I was shown to be involved in the regulation of Ca^+ uptake into SR either by direct phosphorylation of SR Ca 2 + -ATPase (SERCA2) (Toyofuku et al, 1994) or by phosphorylation of phospholamban (PL), which appears to cause dissociation of the inhibitory P L from the S E R C A 2 complex. In the neonatal heart, the lower expression and activity of S E R C A 2 is responsible for the decreased SR release and uptake of C a 2 + (Michalak, 1987). In addition, CaMPKII-phosphorylated P L has only been detected in adult heart (Michalak, 1987). The expression patterns of P K C and C a M P K I I clearly showed that, during development, the heart regulates the expression of these kinases reciprocally (Fig. 7). The down-regulation of P K C , which closely correlated with cell proliferation, may possibly prime cardiomyocytes to the terminal differentiation process. In contrast, the up-regulation of C a M P K may be necessary for adult cardiomyocytes to perform cardiac functions, which require efficient intracellular C a 2 + homeostasis. The up-regulation of C a M P K expression in the heart during development may correlate with the increased demand for Ca2+ homeostasis and the efficient control of myocardial contraction. 6.3. Developmental expression of cyclin-dependent protein kinases and regulation of cyclin-dependent kinase 1 (CDK1) activity. It has been established that the progression of the cell cycle is fastidiously controlled by various cell cycle-dependent protein kinases ( C D K ) . The cardiomyocytes are mitotically active at birth and become terminally differentiated about 2 to 3 weeks after birth (Rakusan, 1984; Zak, 1984a). Even though the molecular mechanism(s) for the terminal differentiation is still unknown, it has become apparent that the cardiomyocytes are biochemically inhibited and do not 197 permanently lose their ability to undergo mitotic cell divisions. Observations of re-initiation of D N A synthesis and mitotic division of adult cardiomyocytes under certain conditions such as pressure overload (Capasso et al, 1993), anemia (Olivetti et al, 1992), T P A treatment (Claycomb and Moses, 1988) and senescent heart in certain strains of rats (Anversa et al, 1991) substantiate this theory. More clearly, the induction of hyperplasia of either the atria or the ventricle by SV40 large-T antigen oncoprotein, which was expressed under the transcriptional control of the promoter regions of either atrial natriuretic factor or a-cardiac myosin heavy chain, strongly support this theory and may provide some insight into the mechanism of terminal differentiation (Katz et al, 1992; Field, 1988). SV40 large-T antigen directly associates with tumor suppressor genes such as the retinoblastoma protein (Rb) or related proteins (p53, p l07 and pl30) (Daud et al, 1993) and it inhibits their activities by sequestering them (McGi l l and Brooks, 1995). The regulation of Rb is important for cells to progress from G I to S phase, and it is regulated by C D K s in normal conditions (Daud et al, 1993). Phosphorylation of Rb by C D K s prevents Rb from interacting with E2F, a transcription factor that targets many genes required during the S phase such as cyclins A and E (Nevins, 1992). In fact, terminally differentiated myotubes could re-enter the cell cycle by SV40 T- antigen through inducing C D K 1 (also referred to as cdc2), C D K 2 , and their partner cyclins A and B (Okubo et al, 1994). More recently, the expression of muscle specific genes was sufficiently inhibited by expression of cyclin D l alone or expression of D l with C D K 2 and cyclin A or E during skeletal myogenesis (Guo and Walsh, 1997). The expression of Rb and other tumor suppressors have been investigated in the heart (Tarn et al, 1995; K i m et al, 1994). It has been shown that p53 and p l07 were only detected in neonatal ventricle, whereas Rb was present in adult and neonatal rat heart. But Rb was highly phosphorylated only in undifferentiated neonatal heart (Tarn et al, 198 1995). In concert with the hyper-phosphorylation of Rb, these authors reported that C D K 1 and 2 were detected in neonatal heart but not in adult heart (Tarn et al, 1995). The expression of C D K partners, cyclins, were previously investigated (Yoshizumi et al, 1995). Based on Northern and Western blots, they have reported that cyclin A was only expressed in under 2 day-old hearts but not in 14 days or older hearts, and cyclin B was detected in both neonatal and adult hearts, but at markedly reduced levels in the adult. Other cyclins, such as cyclin C, D I , D2, D3 and E , were present in both stages without detectable changes in m R N A levels in the human heart. Others, however, reported that cyclin D I and D3 also declined with age in the mouse heart based on protein levels (Soonpaa et al, 1996). This discrepancy may indicate the higher protein turnover rate of D I and D3 in adult heart or may result from the differences between species. In any event, the hypophosphorylation of Rb and expressional regulation of isoforms of C D K s and cyclins may well be one of the mechanisms for terminal differentiation of cardiomyocytes. Based on this hypothesis, I further investigated the expressional regulation of a full spectrum of C D K isoforms during postnatal development of rat hearts. Since various C D K s show redundancy in their roles in cell cycle control, it may be possible that C D K s are selectively expressed in tissue and growth vs differentiation specific manner (Krek and Nigg, 1989; L i et al, 1997). C D K 1 typically associates with cyclin A and B l and is required for the G2/M transition in the mammalian cell cycle (Nigg, 1995; M c G i l l and Brooks, 1995a). In C2C12 muscle cells, serum-withdrawal induced differentiation led to decreased expression and phosphotransferase activity of C D K 1 , even though the m R N A was induced transiently (Jahn et al, 1994). The decrease in activity may in part be due to the increased expression of C D K inhibitors such as p l 6 w i ( 4 and p 2 i c l p l / W A F 1 (Parker et al, 1995b). These results 199 implicate a crucial role for C D K 1 in muscle cell differentiation. C D K 2 in association with cyclin E and A is essential for Gi/S transition and progression through the S phase, respectively. C D K 4 complexes with D-type cyclins and plays a key role in Gi progression. Rb is phosphorylated predominately by CDK4/cycl in D , and probably by CDK2/cycl in E and C D K l / c y c l i n B (Taya, 1997), which appears to be critical for proliferation of myotubes and cardiomyocytes. The role for C D K 5 in cardiomyocytes is not known. In neurons, it associates with the cyclin-like p35 and is implicated in neurofilament phosphorylation in postmitotic neurons (Tsai et al., 1994; Lew et al., 1994; Nigg, 1995). In M L - 1 human myeloblastic leukemia cells, increased CDK5/cycl in D l expression was correlated with decreased D N A syntheis and differentiation (L i et al, 1997). The expression of C D K 1 , C D K 2 and C D K 4 declined rapidly to almost undetectable levels by 20 days of age (Fig. 8). Similarly, the phosphotransferase activity of C D K 1 toward histone H I was also decreased by 20 days after birth (Fig. 11). The expression of C D K 5 , however, diminished more gradually and was detectable even after 50 days of age (Fig. 8L). These results imply that the regulation in the expression of C D K 1 , C D K 2 and C D K 4 are intimately involved in the terminal differentiation of cardiomyocytes. However, the precise role of C D K 5 in heart still remains uncertain and requires further investigation. The role of C D K 8 has not been investigated as extensively as other C D K s , but it appears to be involved in basal transcription and D N A repair rather than in cell cycle regulation (Maldonado et al, 1996). It complexes with cyclin C and phosphorylates the C-terminal domain of R N A polymerase II, which is thought to be involved in the promoter clearance by the polymerase (Fisher, 1997). The presence of C D K 8 throughout the postnatal development of the heart (Fig. 9) is consistent with the putative role of C D K 8 in D N A repair and basal transcription 2 0 0 in ventricles. K K was originally classified as a cyclin-dependent kinase at the time it was identified by cloning studies (Meyerson et al, 1992). Even though it contains two putative phosphorylation sites (Thr-160 and Tyr-162) in a T D Y motif found between subdomains VI I and VIII (Meyerson et al, 1992), which are common to M A P kinases, the activation of K K by E G F does not appear to require these sites to be phosphorylated (Taglienti and Davis, 1996). There has been little information about the role of the K K protein. Western blot analysis indicated that the expression of K K was up regulated with age (Fig. 9F) and it was primarily expressed in brain, heart, kidney, liver, and skeletal muscle (Fig. 9G). The presence of this protein in brain was previously reported based on Western blots and immunoprecipitation (Yen et al, 1995). They observed the expression of three to four new isoforms of K K with apparent molecular masses ranging 40-52 kDa in S D S - P A G E in adult brain, whereas only one isoform, 48 kDa, was detected in fetal brain. In the heart, the three isoforms also immunoreacted with the K K N -terminal antibody. Since among the three isoforms only the 42-kDa protein immunoreacted with the K K C-terminal antibody, this may indicate that these isoforms differ in the C terminus with common N termini. Nonetheless, the increase in the expression of K K isoforms during postnatal development of the heart may indicate a specific role of K K in adult stage, maybe in the differentiated cells. In contrast to my study, Taglienti and Davis (1996) could not detect K K m R N A in the heart. The reason for discrepancy is not clear but it may be due to different isoforms that were targeted for analysis or this protein might be up-regulated via reduced protein degradation rather than an increased m R N A level in the heart. 201 At present, there is little information regarding the role of C D K 7 and W e e l in the heart. C D K 7 , once identified as a C D K activating kinase ( C A K ) , is a subunit of transcription factor IIH (TFIIH) (Drapkin et al, 1996). TFI IH is a multi-subunit complex required for transcription and for D N A nucleotide excision repair and has three enzymatic activities: as an ATP-dependent D N A helicase, a DNA-dependent ATPase and a kinase specific for phosphorylating the C-terminus of R N A polymerase II. The phosphotransferase activity is carried out by C A K , which is composed of C D K 7 , cyclin H and a 36 kDa assembly factor termed Mat-1 (Tassan et al, 1995; Drapkin et al, 1996). The steady-state m R N A level varied significantly in different cell lines and terminally differentiated tissues (Yee et al, 1995). In this study, the up-regulation in expression of C D K 7 observed during development may be associated with an increased role of TFI IH in adult heart or cardiomyocytes rather than with the mitotic state of cells. Wee l expression in the heart was very low compared to other tissues. These results indicate that the proliferation and terminal differentiation of cardiomyocytes were primarily regulated at the level of C D K expression rather than at their target kinases. 6.4. MAP kinase signalling cascades during the development of rat heart. The present study has demonstrated that there is differential regulation of the Raf isoforms during postnatal development of rat heart. Raf l and RafB were significantly down regulated in the developing heart, whereas no changes were detected in RafA expression. Expression of Raf l and RafA was also detected in isolated ventricular myocytes, but RafB was evident only in non-myocytes (Fig. 13 and 14). The activities of Raf l and RafA followed the same pattern as in the expression levels during postnatal development, which showed a decrease of Raf l with no change in RafA (Fig. 24). The observed down-regulation in activity of Raf l was due to lower 2 0 2 enzyme amounts in adult ventricles (Fig. 24). The decreased Raf i activity correlated with down-regulation of the basal specific enzyme activities of E r k l and Erk2, which he downstream of Raf i in the M A P kinase signalling pathway (Fig. 24). The significance of isoform-specific regulation of expression and activity is unclear presently; however, these isoforms have been shown to have distinct distributions and discrete activation mechanisms. In neonatal ventricular myocytes, Rafi and RafA were detected by Northern and Western blot analysis (Storm et al, 1990; Bogoyevitch et al, 1995b). Both isoforms were also shown to be activated by endothelin-1 and phorbol ester, but only Rafi was activated by aFGF (acidic fibroblast growth factor) (Bogoyevitch et al, 1995b). Recent studies with co-expression of Raf isoforms and casein kinase 2 (CK2) in sf-9 insect cells showed that the (3 non-catalytic subunit of C K 2 directly interacts with RafA but not with other isoforms (Boldyreff and Issinger, 1997; Hagemann et al, 1997). These results indicate that R a f i , RafA and RafB are regulated by different mechanisms, which may be involved in distinct roles in the activation of E r k l and Erk2 during postnatal development of the heart. Tpl2 and Mos are other M A P kinase kinase kinases which may activate E r k l and Erk2 and possibly S A P K , but not p38 Hog (Salmeron et al, 1996). The role and identity of these kinases has not been explored in the heart. Western blot analysis demonstrated the presence of both kinases in isolated ventricular myocytes with increasing amounts in adult rat ventricles (Fig. 13). In view of the high expression of Tpl2 in terminally differentiated granular cells (Miyoshi et al, 1995) and other cells, Tpl2 may play an important role in mamtaining ventricular myocytes in their terminal differentiation. Likewise, studies of Xenopus oocytes have shown that the expression of Mos is required for cells to enter meiosis II from meiosis and cell cycle arrest at 2 0 3 metaphase II of unfertilized eggs (Roy et al, 1996). In addition to expression in germ cells, Mos is expressed in various somatic cells and cell lines (Leibovitch et al, 1991). In skeletal muscle, the expression of Mos increases 20- to 40-fold during postnatal development (Leibovitch et al, 1991). The role of Mos in cardiomyocytes has not been previously explored, but the high expression of Mos is involved in the apoptosis of cells in the S phase and causes irreversible cell cycle arrest in the G i phase of Swiss 3T3 cells (Fukasawa et al, 1995). When these cells were in the G2 phase at the time of Mos expression, the cells completed M phase but failed to undergo cytokinesis, resulting in binucleated cells. Considering the temporal regulation of Mos expression, it may be involved in the cell cycle arrest and bi- or poly-nucleation of adult cardiac myocytes. In fact, the over-expression of Mos causes differentiation of C2C12 myoblasts (Leibovitch et al, 1995). Mos directly phosphorylates M y o D , which induces the heterodimerization of M y o D and E12 proteins, resulting in muscle specific gene expression (Lenormand et al, 1997). Both E r k l and Erk2 are activated by various mitogenic stimuli including angiotensin II, endothelin 1, phenylephrine, adenosine, A T P , fibroblast growth factor and mechanical stress in both neonatal cell cultures and adult heart tissues or isolated cardiac myocytes (Sadoshima et al, 1993; Yamazaki et al, 1993; Lazou et al, 1994; Fig. 27 & 28). Activation of E r k l and Erk2 is carried out by the 45-46-kDa dual specific protein kinases M e k l and Mek2 (Crews et al, 1992). In cardiac myocytes, M e k l is activated by stimuli that activate E r k l and Erk2 (Lazou et al, 1994). In this study, Western blot analysis confirmed the previous observations (Lazou et al, 1994) which showed a decrease in expression of both Erk2 and M e k l during development (Fig. 17). In addition, E r k l levels were also analyzed and showed no changes in expression (Fig. 2 0 4 17). Similar to the observed differential expression of Erk isoforms, Rsk isoforms also showed divergent patterns of expression during development (Fig. 17M). Rsk 1 was down regulated up to 50% (to the same extent seen with Erk2), whereas there were no detectable changes in Rsk2. It is still unclear whether there are differences in regulation and functions of E r k l and Erk2, and R s k l and Rsk2 (Zhao et al, 1996). R s k l and Rsk2 have been implicated both in the meiotic maturation of oocytes and the regulation of glycogen synthesis via protein phosphatase 1 (Blenis, 1991; Sturgill and Wu, 1991). R s k l has been reported to be activated in the heart by various mitogenic stimuli, such as endothelin-1, angiotensin-II or mechanical stress (Sadoshima et al, 1995). However, the role and regulation of Rsk2 in the heart have not been investigated in detail. Several protein kinases have been shown to activate S A P K and p38 Hog but not E r k l and Erk2. These protein kinases include Pak isoforms, M l k 3 , T a k l and M e k k l . The regulation and function of these protein kinases in the heart have not yet been investigated. Among three isoforms of Pak, P a k l and Pak2 were detected in ventricles and isolated cardiomyocytes, and their expression was reciprocally regulated during postnatal development: down-regulation of P a k l and up-regulation of Pak2 (Fig. 15). The expression of P a k l and Pak2 in the absence of Pak3 agrees with previous reports that showed the ubiquitous expression of Pak2, brain- and muscle-specific expression of P a k l , and brain-specific expression of Pak3 (Sells and Chernoff, 1997). Little is known regarding the characteristics of the Pak isozymes and their physiological roles in the developing heart, except that P a k l is activated by hyperosmotic shock in neonatal ventricular myocytes (Clerk and Sugden, 1997). 2 0 5 Western blot with Mlk3 antibody revealed the presence of this protein in whole ventricle and isolated ventricular myocytes of rat heart, and its level of expression increased up to 7-fold during development (Fig. 16). Mlk3 has been shown to be expressed in many tissues and cell lines, and shown to interact with Cdc42 and R a c l (Raima et al, 1996; Teramoto et al, 1996; Fanger et al, 1997a). Little is known about the regulation and function of this kinase, but over-expression in COS-7 cells activates J N K signal cascades, probably by direct phosphorylation and activation of S e k l , with little effects on the E r k l and Erk2 and p38 Hog signalling pathways (Teramoto et al, 1996). M e k k l is another protein kinase that is involved in the S A P K signalling pathway by phosphorylating and activating Sek l (Pelech and Charest, 1995), although the mode of M e k k l activation is presently unclear. To date, two modes of activation of M e k k l have been described: one is the N-terminal truncation of M e k k l by caspase-3 (Cardone et al, 1997) and the other is a binding of M e k k l to Ras and Cdc42/Rac (Fanger et al, 1997a). However, neither of these Mekk activation mechanisms have yet been demonstrated in the heart. Western blot analysis with a Mekk specific antibody revealed a 75-kDa protein that increased in amount during development (Fig. 16). The functional roles and significance of the up-regulation of Mlk3 and M e k k l remain to be established. Sek l expression was also up regulated concurrently (Fig. 18), which may imply a more prominent role for S A P K signalling pathways in the adult as compared to the neonatal heart. S A P K represents another family member of M A P kinases which are activated by various stress-related stimuli, such as heat shock, hyperosmostic conditions, U V radiation, 2 0 6 cyclohexamide, anisomycin, phorbol myristic acetate ( P M A ) , and tumor necrosis factor-oc (TNF-a) (Sugden and Bogoyevitch, 1995; Derijard et al, 1995; Robinson and Cobb, 1997). S A P K isoforms were activated by mechanical stretch, sorbitol and endothelin-1 treatment of cultured neonatal ventricular myocytes (Bogoyevitch et al, 1995; Komuro et al-, 1996) and by ischemia/reperfusion in whole hearts (Bogoyevitch et al, 1996b; Knight and Buxton, 1996). The activity of S A P K is directly regulated by Sek l (SAPK/Erk kinase 1, also known as M K K 4 and J N K K ) , which shares 45 % amino acid homology with M e k l and Mek2 (Derijard et al, 1995). Certain stimuli such as osmotic shock, heat shock, taxol, sodium arsenite, anisomycin, lipolysaccharide, interleukin 1 and T N F - a , most of which also activate S A P K , also lead to activation of p38 Hog (Pelech and Charest, 1995; Bogoyevitch et al, 1996b). Activation of p38 Hog is carried out through phosphorylation on both threonine and tyrosine residues in a motif by the dual specific protein kinases, M k k 3 and Mek6 (Han et al, 1996; Han et al, 1997). Mkk3 is an approximately 35-kDa kinase that shares about 41% of its amino acid sequence with M e k l (Han et al, 1997). In the heart, the p38 Hog signalling pathway is activated by cellular stresses such as hyperosmolarity, ischemia and ischemia reperfusion (Bogoyevitch et al, 1996; Knight and Buxton, 1996; Sadoshima et al, 1996). A downstream target for p38 Hog is M A P K -activated protein kinase 2 (Mapkapk2), which can phosphorylate heat shock protein (hsp) 27 and a transcription factor C R E B (Freshney et al, 1994; Clifton et al, 1996). The phosphorylation of hsp27 and C R E B induced by cellular stresses has been suggested to stimulate polymerization of actin and production of c-fos m R N A , respectively (Landry et al, 1995). Western blot analysis of these protein kinases revealed interesting patterns for their expression during development of the heart. Both Sek l and M k k 3 increased in expression in the developing heart, whereas there were no changes in the amount of S A P K or p38 Hog (Fig. 18 & 207 19). Since both of these signalling pathways are activated by various cellular stresses such as ischemia, their up-regulation during development may play a role in age-specific responses to the stimuli, such as less tolerance to ischemia or hypoxia in adult as compared to neonatal heart. In several non-hemopoietic cells or tissues, the activation of S A P K or p38 Hog has been implicated in cell death. In addition, the activation of p38 Hog has been suggested to be involved in the mechanism responsible for preconditioning of the heart, protecting damage from prolonged ischemia by previous transient ischemia. Presently, little information is available regarding the differences in preconditioning between neonatal and adult heart, but the up-regulated expression of this signalling pathway suggests that it plays a significant role in adult heart. Ventricular tissues are biologically and physically active groups of cells that are constantly exposed to neuronal and hormonal stimuli. During postnatal development, these neuronal and hormonal stimuli may vary, which may also affect the activities of various protein kinases. In this regard, I analyzed the specific phosphotransferase activities of E r k l and Erk2, S A P K and Mapkapk2 (Fig. 21, 22, 23 & 24) during the development of the heart. The specific activities of E r k l and Erk2 were down-regulated, whereas the activities of S A P K and Mapkapk2 were low and did not change significantly. The down-regulation of E r k l (in activity) and Erk2 (in both activity and protein levels) occurred concurrently with the decrease in expression and activity of C D K 1 (Fig. 11). C D K 1 has been known to be a prerequisite for the process of cell cycle and its down-regulation has been involved in the terminal differentiation of muscles. Our observations indicate that, in addition to C D K 1 , down-regulation of the E r k l and Erk2 signalling pathways may also participate in the terminal differentiation of ventricular myocytes. 2 0 8 6.5. Insulin signalling cascades during development of rat heart. During the development of the heart, the glycogen content and dependence on glucose and lactate decreases with age and the normal adult heart predominantly utilizes fatty acids, while glycolysis is inhibited. Since insulin stimulates a wide array of signalling cascades, resulting in increased synthesis of protein, lipid and glycogen and enhanced glucose uptake (Denton and Tavare, 1995), I investigated how insulin-activated protein kinases are regulated during postnatal development of the heart. First, to investigate what protein kinases are activated by insulin, I anayzed several protein kinases which have been shown to involved in insulin actions in other cells or tissues after injecting a bolus dose of insulin through tail vein. Unlike previous studies which utilized high doses of insulin (10 U/kg or more), I used a dose of insulin (2 U/kg) that more closely approximated physiological conditions. Under these circumstances, insulin activated P K B , S 6 K and C K 2 without stimulating the E r k l and Erk2 M A P kinases. These results are consistent with a previous report of insulin treatment in isolated cardiomyocytes that led to activation of PI3K and S 6 K without increasing M A P kinase activity (Lefebvre et al, 1996). A role for M A P kinase signalling cascades in the metabolic actions of insulin has been disputed on several grounds (Denton and Tavare, 1995; White et al, 1998). For example, epidermal growth factor can potently activate M A P kinases without affecting glucose transport or glycogen synthesis (Robinson et al, 1993). Also, a direct introduction of activated Raf i failed to increase glucose transport (Fingar and Birnbaum, 1994). White (1998) further suggested that the M A P kinase signalling pathway is not very sensitive to insulin and usually needs a strong insulin signal, which can achieved by overexpression of the insulin receptor. To further investigate how these protein kinases are regulated during postnatal development, 2 0 9 the expression of insulin-activated protein kinases were analyzed. There were modest reductions in the expression levels of PI3K, P K B and S 6 K after birth during the first year. These reductions in expression in conjunction with decreasing circulating insulin levels and increases in expression of 5'-AMP-activated protein kinase ( A M P K ) during development (Girard et al, 1992; Makinde et al, 1997) may contribute to the adult specific glucose metabolism in the heart. However, in the adult ventricle samples, P K B and S 6 K were still highly expressed (Fig 34, 36 & 37), which indicates that the adult rat heart is still quite responsive to insulin as reflected by the P K B / S 6 K signalling pathway. Likewise, as GSK3-(3 levels were maintained during postnatal development (Fig. 38), inhibition of this kinase by P K B may also mediate some of the actions of insulin in adult heart. Some of the most profound changes in kinase expression during heart development were observed for C K 2 . The known in vitro substrates for C K 2 include over 100 proteins and many of them are involved in cell proliferation (Allende and Allende, 1995). The signalling cascades for C K 2 activation and its precise role are not clear, but it has been proposed that its expression and activity are dependent on the proliferation state of the cell (Ulloa, et al, 1993; Gruppuso and Boylan., 1995; Mestres et al, 1994). While there was a reduction of C K 2 protein and activity that correlated with the cessation of myocyte proliferation after day 10, the adult heart still had one of the highest levels of C K 2 detected in the rat (Fig. 39). This supports additional roles for C K 2 besides control of cell proliferation. 6.6. Regulation of MAP kinases by various stimuli. The present study showed that E r k l and Erk2 were activated by SNP , endothelin 1 (ET1) and 210 adenosine (Fig. 26, 27 and 28). The stress-related M A P kinases, such as Mapkapk2, were activated by SNP, adenosine, anisomycin and sorbitol (Fig. 26, 29 and 30). S N P acts as a N O donor and N O targets many proteins, either directly binding heme-containing proteins, such as soluble guanylyl cyclase (sGC), nitrosation of thiol residues, nitration of tyrosine or oxidizing D N A and proteins (reviewed in Balligand and Cannon, 1997). S N P has been suggested to release N O through a reduction-oxidation reaction (Smith et al, 1990) as shown in the following formula: [Fe(CN) 5 NO]" 2 + H 2 0 =» [Fe(CN) 5 H 2 0]" 2 + N O The activation of sGC elevates cytosolic c G M P level, by converting G T P to c G M P , and results in the activation of c G P K . There are other known receptors for c G M P , which include c G M P -stimulated phosphodiesterase, cGMP-inhibited phosphodiesterase and cGMP-gated ion channels (reviewed in Lincoln and Cornwell, 1993). In the present study, S N P activated both E r k l and Mapkapk2 in isolated cardiomyocytes (Fig. 26). However, the E r k l activation appeared to be independent of the elevation of c G M P level, since the E r k l activation by S N P was not inhibited by O D Q , a sGC inhibitor (Fig. 25). In addition, 8 -Br -cGMP failed to activate E r k l (Fig. 26). In contrast, Mapkapk2 (a direct downstream kinase of p38 Hog) was activated by S N P via an O D Q sensitive pathway and the activation was abolished by SB203580, a p38 Hog inhibitor. These results and the activation of Mapkapk2 by 8 -Br -cGMP altogether support the hypothesis that S N P activates Mapkapk2 via elevating c G M P levels and by activating p38 Hog. To date only limited studies have been performed regarding the role of c G M P or c G P K in the regulation of M A P kinases. N O has been shown to activate E r k l , Erk2, p38 Hog and J N K within 10 min in Jurkat human T-cell leukemia cell line (Lander et al, 1996), but the role of c G M P or c G P K in these activations were not addressed. In mesangial cells, neither 8 -Br -cGMP nor dibutyl c A M P can activate Erk isoforms (Haneda et al, 1996). 211 The physiological function of N O has been controversial. In postcapillary endothelium, S N P induces proliferation by activating Erk isoforms (Parenti et al, 1998), whereas in aortic smooth muscle cells, S N P suppresses proliferation by inhibiting the activation of Erk isoforms stimulated by epidermal growth factor (Yu et al, 1997). Several investigators have reported that SNP, as well as c G M P , induce apoptosis in several cells, such as pancreatic B-cell line, smooth muscle cells and neonatal cardiomyocytes (Loweth et al, 1997; Nishio et al, 1996; W u et al, 1997). However, the role of S N P and c G M P in adult cardiomyocytes still remains to be explored. During cardiac ischemia and ischemia/reperfusion, N O is produced and peaked (more than 10-fold above the preischemic level) at 40 s of reperfusion (Wang and Zweier, 1996). On the one hand, N O may interact with superoxide, resulting in the generation of peroxynitrite and cellular injury. On the other hand, N O is involved in the postischemic cardioprotection, sustaining biomechanical performance independently of endothelial function (Massoudy et al, 1995; Szekeres et al, 1997). In addition, N O has been implicated in triggering the cardioprotective effects that occur 24-76 h after brief ischemia and reperfusion, which is known as the delayed P C (Bolli et al, 1997). Altogether, N O is involved in induction of cellular damage and apoptosis, as well as in cardioprotection against ischemia. These bimodal effects of N O in the heart may be due to its multiple cellular targets. Even though the detailed mechanism of these effects remains to be investigated, it may be worthwhile to note that the apoptotic and cytotoxic effects of N O and c G M P are induced only during prolonged exposure of these agents to cells (24 h or more). However, the production of N O during reperfusion after ischemia peaks in 40 s and gradually decreases to basal level in 15 min (Wang and Zweier, 1996). This short-lived production of N O may be efficient for cardioprotective effects. The activation of Mapkapk2 by S N P and c G M P in 5 min shown in this study (Fig. 26) may be a key signalling pathway in ischemic P C . Therefore, 212 studying the involvement of N O in ischemic P C and the signalling mechanism of the cardiac protection may be important. The possible role of Mapkapk2 in cardiac protection is discussed in Section 6.7. In contrast to the vaso-relaxation effects of N O , ET1 has been implicated in the contraction of various smooth muscles and in the release of endocrine hormones in the pituitary and kidney (Rubanyi and Polokoff, 1994). In the heart, ET1 causes positive inotropic, chronotropic and mitogenic effects (Shubeita et al. 1990; Ito et al, 1991; Sokolovsky, 1993; Bogoyevitch et al, 1994; Rubanyi and Polokoff, 1994; Rankin, 1994; Hilal-Dandan et al, 1997). In the present study, ET1 activated E r k l about 2.5-fold in 5 min (Fig. 27). The mitogenic effect of ET1 has been implicated in the pathology of hypertrophy in cardiomyocytes, in part by activating M e k l and E r k l / 2 (Gillespie-Brown et al, 1995). ET1 receptors, E T R A or E T R B , have been shown to be coupled to various G proteins, including G a s , G a i , G a q and G a i i , resulting in an increase of intracellular C a 2 + concentrations, activation of P K C , activation or inhibition of P K A and activation of protein-tyrosine kinases such as focal adhesion kinase (Jouneaux et al, 1993; Eguchi et al, 1993). In contrast, adenosine decreases the heart rate and contractility (Lester et al, 1996), by inhibiting activation of A C through G«i, and is also involved in the protection of the heart from ischemia (Liu et al, 1991, Cohen et al, 1995; Marala and Mustafa, 1995). 6.7. Regulation of M A P kinases b y adenosine and its role in ischemic preconditioning of the heart. Endogenous adenosine is released during cardiac ischemia as a result of increased breakdown of A T P to A M P and A M P to adenosine (Chen and Gueron, 1996; Chen et al, 1996). It has further 213 been demonstrated that, in cardiomyocyte cultures, anoxic myocytes secreted adenosine in quantities sufficient for adenosine-receptor activation (Ikonomidis et al, 1997). The increased release of adenosine has been suggested to be a key player in the preconditioning (PC) of the heart (Cohen et al, 1996b; Headrick, 1996). The signalling pathway implicated in the cardiac P C remains to be delineated, but in rat and rabbit hearts, the activation of phospholipase D and P K C appears to be important (Bugge and Ytrehus, 1995; Mitchell et al, 1995; Cohen et al, 1996a; Miyawaki et al, 1996). The activation of p38 Hog has also been demonstrated during reperfusion of ischemic hearts and during ischemia of preconditioned hearts (Pombo et al, 1994; Bogoyevitch et al, 1996; Weinbrenner et al, 1997). It has also been shown that SB203580, a p38 Hog inhibitor, is able to block the protection induced by P C . In addition, anisomycin (500 ng/ml) markedly protected cardiomyocytes from ischemic damages (Weinbrenner et al, 1997). As Figure 30 demonstrates, anisomycin (50 ng/ml) also activated Mapkapk2 in the rat heart. Besides adenosine, several other endogenous agonists such as norepinephrine and bradyldnin have been demonstrated to be involved in the P C heart (Tsuchida et al, 1994; Brew et al, 1995; Goto et al, 1995; H u and Nattel, 1995). It is not yet known whether these agonists also activate Mapkapk2, but P K C is commonly activated by these agonists and P K C inhibitors amply inhibit the P C triggered by these agents (Tsuchida et al, 1994; Ikonomidis et al, 1997). The attenuation of c A M P levels is an another event that occurs during ischemia and reperfusion (Sandhu et al, 1996) and is mediated in part by the increase of G i and reduction of Gs (Ravingerova et al, 1995). Treatment with the adenylyl cyclase activator, N K H 4 7 7 , is able to block P C (Sandhu et al, 1997). Since adenosine inhibits A C via activation of Gi, it may be possible that inhibition of A C during P C by adenosine in fact sensitizes the Mapkapk2 signal transduction pathway(s), which wil l then allow 214 the activation of Mapkapk2 during subsequent prolonged ischemia. The physiological role of Mapkapk2 activation in the P C heart is still to be investigated but Mapkapk2 can phosphorylate several transcription factors, such as C R E B , ATF-1 and hsp27 (Tan et al, 1996). Unphosphorylated hsp27 acts as an actin cap-binding protein in the cell and inhibits polymerization of F-actin filaments. Thus, the phosphorylation of hsp27 by Mapkapk2 appears to stimulate the polymerization of actin, and this is thought to facilitate recovery of actin microfilament networks, which are disrupted during cellular stresses (Landry and Huot, 1995; Guay et al, 1997). Huot et al. (1996) showed that cells overexpressing wild type hsp27 were more resistant to injury from oxidative stress than cells overexpressing an mutated hsp27 that lacks the phosphorylation site. Huot et al. (1997) further demonstrated that the phosphorylation of hsp27 was specifically mediated by the p38 Hog and Mapkapk2 pathway. In the heart, adenosine influences cardiac function either by directly stimulating the adenosine receptors A i and A 3 in the cardiomyocytes or via stimulating A2 receptors located in the systemic vasculature (Collis, 1989; Finegan et al, 1996; Auchampach et al, 1997). The cardioprotective effects of adenosine during ischemia may result from both a vasodilation of coronary vessels and a reduction of neutrophil infiltration via A2 receptors, but adenosine also affects the metabolism of glucose and may provide the cardioprotective effects directly to cardiomyocytes during ischemia (Finegan et al, 1996; Auchampach et al, 1997; Matherne et al, 1997). Adenosine inhibits glycolysis and increases glucose oxidation, resulting in the reduction of proton production (Finegan et al, 1996a). The reduction of proton production is related to the improved recovery of mechanical function during reperfusion of the heart. However, the beneficial effects of adenosine are reversed when adenosine is applied to the preconditioned heart (Finegan et al, 1996b). In other 215 words, adenosine increases glycolysis and proton production, resulting in a depression of mechanical work of the heart. However, these effects are not mediated by adenosine A l receptors, since N6-cyclohexyladenosine, an A l specific activator, still inhibits glycolysis and proton production. It is not known in what mechanism adenosine regulates the metabolism of glucose in control and preconditioned hearts. As discussed earlier, the neonatal heart has been reported to be more tolerant to hypoxia or ischemia than the adult heart. Adenosine was suggested to be involved in the hypoxia-induced coronary vasodilation and the decrease of adenosine sensitivity of aortic rings in adult heart may cause less tolerance to hypoxia. Blockade of adenosine receptors reduced vasodilation and abolished the enhanced tolerance of immature hearts to hypoxia (Matherne et al, 1996). The decreased sensitivity of the adult heart to adenosine may have resulted from the decrease in expression of Gi with no change in G s isoform with age (Hansen, 1995; Bartel et al, 1996). However, it still remains to be investigated how heart development influences the activation of E r k l and Erk2 and Mapkapk2 by adenosine. Even though there have as yet been no direct studies showing Gi-mediated activation of E r k l and Mapkapk2 by adenosine, it is possible that G i and G(3y activate small G proteins such as Ras and Rac, which can in turn activate Raf and Pak isoforms (Bokoch, 1996; Post and Brown, 1996). In this case, the activation of E r k l and Mapkapk2 by adenosine is expected to be decreased during postnatal development of the heart. In contrast, I showed that during development, the expression of M k k 3 and Sek l increased. How these changes may affect the regulation of Mapkapk2 and S A P K by adenosine during development remains to be investigated. 216 E r k l appears to be activated transiently during ischemia and ischemia followed by reperfusion and the activation is probably due to the production of free radical such as peroxide or peroxynitrite (Wang and Zweier, 1996; Guyton et al, 1996; Abe et al, 1998). Recently, it has been reported that E r k l and Erk2 were translocated to the nucleus during ischemia and the activated Erk2 in the nucleus was detected only after reperfusion (Mizukami and Yoshida, 1997). They also suggested that activation of Erk2 is mediated by the activation of Mek2, since Mek2 was rapidly translocated to the nucleus and activated during reperfusion. The activation of E r k l and Erk2 may play key roles in the induction of hypertrophic marker genes and re-initiation of mitosis after ischemia and reperfusion of cardiomyocytes. 6.8. Phosphoproteins during development of rat heart ventricle and identification of a novel histidine protein kinase. Reversible protein phosphorylation is a ubiquitous cellular mechanism that regulates the function of enzymes, ion channels, transcription and translation factors and other proteins in response to extracellular stimuli. Besides the known protein kinases studied in the previous sections, possible novel protein kinases or their substrates can be readily identified as in vitro phosphorylating proteins by incubating [y- 3 2 P]ATP with crude or partially purified extracts. Although, this experimental approach is limited in its specificity (since there are number of non-protein kinases which can become transiently phosphorylated), it can be a useful initial step for identifying a novel protein kinase or a substrate. The phosphotransferase activities can further be compared and quantified in response to various cell responses. A phosphoprotein of interest can then be purified and used for protein sequence analysis. Using this method, at least 8 phosphoproteins were detected that underwent changes during the postnatal development of rat 217 heart ventricles (Fig. 43). The change in detected P incorporation into proteins may be explained by changes in either activity and/or expression of the responsible kinases or the activities of phosphatase during postnatal development. The identities and roles of these phosphoproteins are still to be determined; however, a 40-kDa phosphoprotein was characterized in more detail and may prove to be a novel mammalian histidine protein kinase. The evidence that this 40-kDa protein is a histidine protein kinase is divided into two categories: First, the 40-kDa protein was phosphorylated on histidine residue(s) and second, the protein appeared to be a kinase. The first piece of evidence is based on the observations that the phosphate linkage was sensitive to boiling in neutral or acidic conditions, while there were no effects with boiling under basic conditions. The phosphate linkage was also unstable in the presence of 18% pyridine (Fig. 45). More conclusively, basic phosphoamino acid analysis verified the presence of phosphohistidine in the 40-kDa phosphoprotein (Fig. 51). Data supporting the second piece of evidence are the molecular determination of the histidine protein kinase autophosphorylation following gel filtration and the co-migration of azido-ATP label with the 40-kDa protein in S D S - P A G E gels and 2-dimensional electrophoresis (Fig. 50). The protein retained phosphorylation activity even after sequential column purification steps, which additionally supported its identification as a kinase. At the same time, this kinase was unlikely to be an ATPase which catalyses reactions via phospho-enzyme intermediates, since it was mainly localized in cytosolic fractions (Fig. 42), was also sensitive to temperature in phosphotransfer kinetics (Fig. 48), and its autophosphorylating activity was inhibited by genistein (Fig. 45 and 46) which is a known tyrosine and histidine protein kinase inhibitor and D N A topoisomerase inhibitor (Huang et al, 1992; Constantinou et al, 1990; Okura et al, 1988; Markovits et al, 1989). 218 The histidine protein kinase appeared to be distinct from the 36- to 38-kDa histidyl phosphoprotein, previously observed in rat liver extracts (Motojima and Goto, 1993; Hegde et al., 1993; Motojima and Goto, 1994). That 38-kDa protein was primarily found in plasma membrane fractions, was insensitive to genistein, eluted at 70-75 kDa by gel-filtration and underwent phosphorylation maximally by 4 min at 20 °C and 0 °C (Hegde, et al, 1993; Motojima and Goto, 1994). In contrast, the 40-kDa histidine protein kinase in the present study was detected primarily in the cytosolic fraction, was sensitive to genistein, eluted at 40-kDa size in gel-filtration and displayed linear phosphorylation kinetics for at least 45 min at 30 °C, which was 10 times lower at 0 °C. The effect of genistein was also observed for histidine protein kinase from yeast (Huang, et al, 1992). A simple linear log-dose relationship for inhibition was observed and 50% inhibition was achieved with 2 m M of genistein (Fig. 46B). The reported IC50 values for genistein varied from 110 p M to 1 m M depending on the proteins (Constantinou et al, 1990, Huang, etal, 1992). The role of this kinase in the heart has not yet been studied and awaits protein sequence data for cloning. This kinase was highly activated only after 10 days following birth (Fig. 44) and was most evident in the heart, with much less being found in skeletal muscle and testes (Fig. 52). Even though it is not known whether the increased autophosphorylating activity was due to an increase in specific autophosphorylating activity or an increase in protein expression, the higher activity associated with development may indicate a possible role of this kinase in the development of muscles. In prokaryotic and lower eukaryotic cells, several genes that encode histidine protein kinases have been cloned and their proteins purified (Huang, et al., 1991). 219 However, histidine protein kinases have neither been purified nor cloned from a mammalian system. The 40-kDa kinase identified in this study is the first member of a mammalian kinase family that autophosphorylates at a histidine residue(s) by forming N-linked protein phosphorylation. 2 2 0 7. SUMMARY AND CONCLUSION The rat heart ventricle expresses several protein kinases in an age-specific manner, many of which may be involved in distinct physical and biochemical processes during postnatal development. The expression of cyclic nucleotide-dependent protein kinases, c A P K and c G P K , were slightly decreased or did not change. In contrast, those protein kinases closely involved in cell proliferation and cell division, such as P K C s and some C D K s (including C D K 1 , 2, 4 and 5) were down-regulated rapidly after 10 days of age, which may well act to keep cardiomyocytes terminally differentiated. Other C D K s such as C D K 7 , W e e l , C D K 8 and K K , however, did not change or rather increased, which may indicate that C D K 7 and 8 are involved in house keeping functions, such as basal transcription and D N A repair and K K in terminal differentiation of the cell. Expression of mitogenic protein kinases including R a f i , RafB, M e k l , Erk2 and R s k l were significantly down-regulated, whereas the stress signalling kinases, such as M l k 3 , M e k k l , S e k l , M k k 3 and Mapkapk2 were up-regulated during postnatal development. The activities for Ra f i , E r k l , Erk2 and R s k l were decreased during development, whereas no changes were detected in RafA and Rsk2. However, the specific activities changed only for E r k l and Erk2 with age, which was concurrent with the decrease in C D K 1 activity. The decrease in expression and activities of these mitogenic signalling pathway components may be an important step for cells to undergo differentiation and to keep them in a differentiated state. The proto-oncogene-encoded kinases Mos and Cot/Tpl2 were up-regulated up to 2- and 4-fold, respectively, during postnatal development, but their roles in the heart are not known. 221 Since these kinases are highly expressed in differentiated cells, they may be involved in the cessation of cardiomyocyte proliferation. There were modest reductions in the expression of PI3K, P K B and S 6 K after birth during the first year, but even in the adult ventricles, P K B and S 6 K were still highly expressed. Therefore, the adult heart was still quite responsive to insulin and this was achieved in part through the P K B / S 6 K signalling pathway. Likewise, as GSK3(3 levels were maintained during postnatal development, inhibition of this kinase by P K B may also mediate some of the actions of insulin in the adult heart. Some of the most profound changes in kinase expression during heart development was observed for C K 2 . The known in vitro substrates for C K 2 include over 100 proteins and many of them are involved in cell proliferation. The signalling cascades for C K 2 and its precise role are not clear but it has been proposed that its expression and activity are dependent on the proliferation of cells. While there was a reduction in C K 2 protein and activity that correlated with the cessation of myocyte proliferation after day 10, the adult heart still had one of the highest levels of C K 2 detected in the rat. This supports additional roles for C K 2 besides control of cell proliferation. Studies with various activators for c A P K , c G P K and P K C in isolated cardiomyocytes showed that P K C lies upstream for E r k l and Erk2, whereas c G P K mediated activation of p38 Hog and Mapkapk2. c A P K failed to exhibit effects on either of the two kinases. Similary, SNP, a N O producer, activated E r k l and Mapkapk2 in cGMP-independent and -dependent pathways, 2 2 2 respectively. The signalling pathways for the activation of Mapkapk2 by c G P K have not yet been reported in the literature. ET1 , which is involved in the proliferation and hypertrophy of cardiomyocytes, activated E r k l and Erk2. Adenosine, a mediator of preconditioning of the heart, activated E r k l , Erk2 and Mapkapk2 (2-fold). Insulin, which regulates glycogen metabolism, activated P K B , S6K and C K 2 , whereas E r k l and Erk2 were not stimulated under these conditions. The investigation for potential protein kinases or their substrates has revealed at least 8 phosphoproteins that undergo change during development of the heart. Among them, a 40-kDa protein may be an autophosphorylating histidine protein kinase, in which case it would be the first identified histidine protein kinase in a mammalian system. This putative histidine protein kinase was sensitive to genistein and highly active in the terminally differentiated tissues, especially in the adult hearts which demonstrated the highest activity among eleven different rat tissues tested. 2 2 3 FUTURE STUDIES Advances have been made by this study in our understanding of the extent of protein kinase expression and activity during postnatal development of the rat heart ventricle. The identification and preliminary characterization of what is potentially a novel mammalian histidine protein kinase was another contribution made from this thesis research. At the same time, many questions were also raised, which need to be addressed in further studies. These include: 1. What is the significance in the change in expression and activities of protein kinases during development? Protein kinases, which undergo significant changes in expression during development, may play age-specific roles in the heart. However, other factors such as changes in the localization of the kinase, the concentration of specific phosphatases or inhibitors, and/or other competing signalling components should be considered at the same time while interpreting these data. To address this point, specific agonists for a distinct kinase or pathway can be used in order to compare the effects of kinases in neonatal and adult ventricles. 2. What are the roles of Tpl2 and Mos that were up-regulated in expression during development? To date no adequate biochemical tools are available to measure the endogenous activities of Tpl2 and Mos. Molecular and genetic studies in the neonatal cardiomyocytes culture, for example, involving over-expression or mutation of Tpl2 and Mos , may indicate their physiological roles during development. 2 2 4 3. Even though the ventricle is mainly composed of ventricular myocytes (up to 75-80% of total cell volume), the tissue is still heterogeneous. Therefore, observations detected in the ventricular tissue may not truly represent the changes in ventricular myocytes. The ventricular myocytes can be isolated from adult as well as from neonatal heart and compared accordingly. 4. What is the significance in the isoform specific expression of kinases, such as P a k l vs Pak2, Raf i vs RafA, E r k l vs Erk2, and R s k l vs Rsk2? There has been no documentation regarding the isoform-specific roles of Pak, Raf, Erk and Rsk in the heart. The activities of these kinases were closely related to the expression levels. B y treating heart or ventricular myocytes with various agonists, specific regulation mechanisms for these kinase isoforms can be uncovered. 5. The two peaks of hsp27 phosphotransferase activity resolved by Resource Q column were increased in the adenosine-treated heart or during ischemia of the P C heart. One of these peaks co-eluted with Mapkapk2, whereas the identity of the kinase in the other peak is still unknown. At this moment, which of these kinases are regulated by p38 Hog is unknown. This issue can be addressed by using p38 Hog inhibitor, SB203580, followed by Resource Q fractionation and kinase assays. The hsp27 kinase responsible for the peak may also be identified by "in-gel" kinase assays, and further purified and characterized. 6. What is the mechanism for c G M P in activating p38 Hog signalling pathway? The physiological significance of cGMP-induced p38 Hog activation and the identification of physiological targets remain to be explored. 225 7. What are the identities and roles of the phosphoproteins that undergo developmental changes? At least 8 phosphoproteins were detected from crude in vitro labeling assays. These phosphoproteins could be further resolved by 2-dimensional electrophoresis, followed by tryptic digestion of the protein. Peptides digested from these enzymes can be subjected to electrospray mass spectrometry analysis and possibly identified by comparisons of the change to mass ratios with predicted tryptic peptides from protein sequence data bases. At the same time, the phosphoproteins can be further purified by column chromatography and ultimately subjected to protein microsequencing. 8. The identification, preliminary characterization and partial purification of a putative novel histidine protein kinase could allow the elucidation of novel signalling pathways as yet unexplored in mammalian systems. 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Resource S column chromatography. Elut ionno. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 40 k D a -268 

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