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Purification and characterization of an insulin-stimulated kinase Zhande, Rachel 1991

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PURIFICATION AND CHARACTERIZATION OF AN INSULIN-STIMULATED KINASE. by RACHEL ZHANDE DIPLOM INGENIEURIN CHEMIE TECHNISCHE FACHHOCHSCHULE, BERLIN 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November 1991 © Rachel Zhande, 1991  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.  (Signature  Department of ^ The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ^C3e-e. (R 191  ii  ABSTRACT  The rapid effects of insulin action appear to be mediated at least in part by the activation of a number of discrete protein serine/threonine kinases. The cellular targets of some of these kinases include key metabolic enzymes such as acetyl-CoA carboxylase (ACC) and the ribosomal S6. While, the S6 kinases have now been purified and characterized, very little is known about the nature of the protein kinase(s) able to phosphorylate ACC particularly on the insulin-directed site. Previous studies demonstrated that a myelin basic protein-kinase (MBP-kinase) from sea-star is able to phosphorylate the insulin-directed site on ACC. Thus, it was interesting to find out whether a mammalian homolog of this kinase existed. Rapid chromatography of supernatant fractions from rat adipose tissue by MonoQ ion exchange using fast protein liquid chromatography revealed several peaks of insulin-stimulated protein-serine/threonine kinase activity towards myelin basic protein. Progress in the purification and characterization of these kinases was initially impeded by the rapid decay in protein kinase activity of these extracts. Thus, a major concern initially was stabilization of the insulin-stimulated protein serine/threonine kinase activity which would enable subsequent purification and characterization of the kinases. Stabilization was achieved by the use of phosphatase inhibitors, particularly in combination with a rapid  iii  procedure which included ammonium sulphate precipitation. This enabled storage of the activated kinases (with apparently very little loss in activity) and further purification. After first examining the chromatographic properties using individual column techniques, the insulinactivated serine/threonine kinases were purified by a procedure which involved ammonium sulphate precipitation, and sequential chromatography on polylysine-agarose, MonoQ, heparin-agarose and a final MonoQ. The purified enzyme showed only two major silver-stained polypeptides (with subunit sizes of 200 and 44 kDa) as judged by SDS-PAGE. From analysis of Western blots performed with several different anti-kinase antibodies, the 44 kDa polypeptide was found to be immunologically related to a sea-star MBP-kinase as well as to a family of mammalian mitogen-activated kinases designated ERKs. It is concluded that the 44 kDa MBP-kinase in rat adipose tissue is a member of the family of mitogen-activated kinases. However, the precise relationship to these kinases as well as the regulatory properties of this insulinactivated kinase await further characterization including molecular cloning of the rat adipose enzyme.  iv TABLE OF CONTENTS  ii vi vii viii ix 1 INTRODUCTION ^ 1.1 Rapid Metabolic Effects of Insulin ^ 1 1.2 Characteristics of the insulin receptor ^ 3 1.2.1 Structure of the receptor ^ 3 1.2.2 Receptor tyrosine kinase ^ 4 1.3 Signalling mechanisms implicated in the mechanism of insulin action ^ 5 1.3.1 Protein phosphorylation/dephosphorylation (in insulin action) ^ 5 1.3.2 Phospholipid (in insulin action) ^ 7 1.3.3 Glycosyl phosphatidylinositol (in insulin action) ^ 9 1.4 Insulin promotes increases and decreases in specific protein-serine/threonine 10 phosphorylation ^ 1.5 Insulin-stimulated protein serine/threonine 16 kinases ^ 18 1.5.1 S6 kinases ^ 1.5.2 p42 and p44 MAP (Mitogen-activated protein) 21 kinases ^ 1.5.2.1 Activation of 90 kDA S6 kinases by p42 23 MAP kinase ^ 25 1.6 The thesis investigations ^ 27 MATERIALS AND METHODS ^ 27 1.1 Materials ^ 1.2 Methods ^ 28 1.2.1 Tissue incubation and preparation of 28 extracts ^ 1.2.2 Ammonium sulphate precipitation ^ 29 1.2.3 Assay of kinase activity ^ 30 1.2.4 SDS-Polyacrylamide gel electrophoresis ^ 31 1.2.5 Silver staining of gels ^ 32 1.2.6 Autoradiography ^ 32 1.2.7 Scanning Densitometry ^ 33 1.2.8 Chromatography ^ 33 1.2.8.1 DEAE-cellulose ion exchange ^ 33 1.2.8.2 Phenyl-sepharose chromatography ^ 34 1.2.8.3 MonoQ ion exchange. ^ 34 1.2.8.4 Affinity chromatography using MBPAffigel column ^ 35 1.2.8.4.1 Coupling of. MBP to Affigel ^ 35 1.2.8.4.2 MBP column chromatography ^ 36 1.2.9 Western blotting Analysis ^ 36 1.2.9.1 SDS-PAGE and Transfer of proteins ^ 36 1.2.9.2 Antibody binding ^ 37 1.2.10 Immunoprecipitation ^ 38 ABSTRACT ^  List of Figures ^ List of Tables ^ List of Abbreviations ^ Acknowledgements ^  V  1.2.11 Protein Assays ^  RESULTS ^  39 40  1.0 Instability of protein kinase activity in extracts of rat fat pad tissue ^ 40 2.0 Stabilization of insulin-activated kinases ^ 43 2.1 Effect of phosphatase inhibitors ^ 43 2.1.1 Estimation of phosphatase activity in crude extracts of rat adipose tissue ^ 44 2.1.2 Effects of microcystin on the phosphorylation of myelin basic protein by protein kinases present in rat adipose tissue ^ 45 2.2 Ammonium sulphate precipitation ^ 48 3.0 Purification of insulin-activated protein serine/threonine kinases from rat adipose tissue ^ 50 3.1 Application of various chromatographic techniques for purification of insulinactivated serine/threonine kinases from rat adipose tissue ^ 53 3.1.1 MonoQ chromatography. ^ 53 3.1.2 DEAE-cellulose chromatography ^ 55 3.1.3 Phenyl-sepharose chromatography ^ 57 3.2 Purification of insulin-activated protein serine/threonine kinases using sequential chromatography with polylysine-agarose, monoQ, heparin-agarose and monoQ ^ 59 4.0 Western blotting analyses ^ 65 4.1 Immunoblottinq with antibody raised against sea-star p44 mPt ^ 66 4.2 Immunoblotting with R2 antibody ^ 73 4.3 Immunoblotting with R1 antibody ^ 73 4.4 Immunoblotting with anti-phosphotyrosine antibody ^ 74 4.5 Immunoblotting with GEGA antibody ^ 74 5.0 Additional characterization of rat adipose protein serine/threonine kinases ^ 76 5.1 Inhibition of endogenous protein phosphorylation by added myelin basic protein ^ 76 5.2 Immunoprecipitation of MBP-kinase activity ^ 78 DISCUSSION ^ 79 Stabilization of insulin-activated MBP-kinases ^ 81 Purification of insulin-activated MBP-kinases ^ 82 Relationship of adipose tissue p44 MBP-kinase to other insulin-stimulated protein serine/threonine kinases ^ 84 Inhibition of endogenous protein phosphorylation by added myelin basic protein ^ 88 Immunoprecipitation ^ 89 Time course/Dose response for MBP-kinase activation ^ 90 Conclusion ^ 92  vi REFERENCES ^  93  LIST OF FIGURES  Figure la. Effect of microcystin on phosphorylation of MBP by protein kinases in high speed supernatant fractions ^  46  Figure lb. Effect of microcystin on phosphorylation of myelin basic protein ^  47  Figure 2  MonoQ chromatography of insulin-activated serine/threonine kinases from rat adipose tissue 54  Figure 3  DEAE-cellulose ion exchange chromatography ^ 56  Figure 4  Phenyl-Sepharose chromatography ^ 58  Figure 5  Polylysine-agarose chromatography ^ 61  Figure 6  MonoQ ion exchange chromatography ^ 62  Figure 7  MonoQ ion exchange chromatography ^ 63  Figure 8  SDS-PAGE of purified insulin-stimulated protein serine/threonine kinase ^ 64  Figure 9  Chromatography of insulin-stimulated serine/threonine kinases on MonoQ and immunoblotting with various antibodies ^ 67  Figure 10  Immunoblotting of insulin-activated kinases partially purified on polylysine-agarose ^ 70  Figure 11  Immunoblotting of purified insulin-activated protein serine/threonine kinases ^ 71  Figure 12  Immunoblotting of fractions from polylysineagarose peak 2 ^  72  Figure 13  Inhibition of endogenous protein phosphorylation by added MBP ^  77  vii LIST OF TABLES  Table 1  Proteins exhibiting changes in phosphorylation in response to insulin ^ 12  Table 2  Insulin-sensitive serine/threonine kinases ^ 17  Table 3  Loss of insulin-stimulated kinase activity under different storage conditions ^ 42  Table 4  Precipitation of myelin basic protein-kinase ^ 49  Table 5  Various chromatographic matrices used in attempts to purify insulin-stimulated protein serine/threonine kinases ^  52  viii LIST OF ABBREVIATIONS  ACC^acetyl-CoA carboxylase ATP^adenosine triphosphate cAMP^cyclic adenosine 3',5'-monophosphate DEAE-cellulose^diethyl-amino-ethyl cellulose DTT^dithiothreitol EDTA^ethylenediaminetetraacetic acid EGTA^ethyleneglycoltetraacetic acid ERK^extracellular signal-regulated kinase Fig.^figure FPLC^fast protein liquid chromatography HSS^high speed supernatant from rat adipose tissue. MPK^meiosis-activated protein kinase MAP^microtubule-associated protein MAPK^mitogen-activated protein kinase MBP^myelin basic protein MOPS^3-[N-morpholino]propanesulfonic acid inorganic phosphate P PAGE^polyacrylamide gel electrophoresis PMSF^phenylmethylsulfonylfluoride RSK^ribosomal S6 kinase SDS^sodium dodecyl sulfate TCA^trichloroacetic acid Tris^tris(hydroxymethyl)aminomethane UNITS OF MEASUREMENT  Da^ DPM^ g xg^ k^ 1^ m^ M^ mol^ A^ min^ n p^  dalton disintegrations per minute gram times gravity kilo liter milli molar mole micro minute nano pico  ix AKNOWLEDGEMENTS  The final work may bear one person's name but it is almost always the result of collaborative efforts. This is certainly true with this thesis. A number of people have shared their insights, ideas and experiences with me and I would like to thank all of them. A very special thanks to Dr. R.W. Brownsey for the excellent supervision, constant encouragement and support during the course of this study. Many thanks to Drs. S.L. Pelech and J.S. Sanghera for their assistance in chromatography work as well as the supply of antibodies. Also, the generous gifts of antiphosphotyrosine antibodies and myelin basic protein from Alice Mui and Frank Nezil, respectively, are greatly appreciated. I also wish to extend my gratitude to Dr. Kath Quayle and Bob Winz and Ed Hanada for the support advice and technical help offered. Finally, the Commonwealth University Scholarship received is gratefully acknowledged.  1  INTRODUCTION.  This thesis is concerned with studies designed to further our understanding of the biochemical basis of insulin action. In particular, the studies are concerned with rapid metabolic responses of cells observed within a few minutes after exposure to the hormone.  1.1 RAPID METABOLIC EFFECTS OF INSULIN.  The hormone insulin regulates a wide spectrum of metabolic processes in a variety of mammalian tissues (Czech, 1977; Denton, 1986). Of particular importance is the modulation of metabolic pathways leading to increased synthesis of protein, glycogen and fat. Concomittantly, the rates of the opposed, catabolic pathways of gluconeogenesis, lipolysis and glycogenolysis are coordinately inhibited in response to insulin. The signaling system or systems which mediate these responses are initiated by a membrane-bound insulin receptor and result in the regulation of key controlling enzymes in their respective metabolic pathways. In view of the wide range of changes in metabolism induced by exposure of different cell types to insulin, a correspondingly large number of possible experimental systems may be considered appropriate to investigate the mechanism of action of the hormone. Indeed, a vast literature has developed describing studies of insulin action and the references quoted here are necessarily  2  restricted in scope. The thesis goals and the introduction here have been motivated by earlier studies of insulin action in rat adipose tissue, which has been an important model system experimentally. In this tissue, effects of insulin may be divided into those effects which can be determined upon exposure of cells to insulin alone (such as stimulation of fatty acid biosynthesis) and alternatively, effects which are most apparent in the presence of hormones which increase cellular cyclic AMP concentration (such as inhibition of catecholamine-stimulated lipolysis). These latter effects are likely to be even more complex than direct effects of insulin alone and will not be described in detail here. Of the direct effects of insulin acting alone, the stimulation of fatty acid biosynthesis for example, involves increases in glucose transport (Simpson and Cushman, 1986), activation of enzymes including pyruvate dehydrogenase (Coore et al., 1971) and acetyl-CoA carboxylase (Denton et al., 1981; Halestrap and Denton, 1973; Lee et al., 1973).  These changes appear to be brought about by alterations in specific activity rather than protein synthesis or degradation and are still apparent following tissue disruption and subcellular fractionation.  3  1.2 CHARACTERISTICS OF THE INSULIN RECEPTOR.  1.2.1 STRUCTURE OF RECEPTOR.  The insulin receptor is an integral membrane glycoprotein with an apparent relative molecular mass (Mr) of 350-400,000 composed of two a-subunits (Mr 130,000) and two B-subunits (Mr 95,000). The a and B subunits are held together by strong disulfide bridges. The insulin binding domain is located on the a-subunit which is completely extracellular. The B-subunit possesses the single transmembrane domain and a cytoplasmic region containing a protein-tyrosine kinase domain (including the putative ATP binding site) and tyrosine phosphorylation sites. It is the B-subunit which is autophosphorylated on tyrosine residues upon the binding of insulin to the a-subunit. This autophosphorylation in turn activates the protein-tyrosine kinase activity of the insulin receptor, rendering the receptor capable of phosphorylating other substrates. The insulin receptor is therefore multifunctional; it contains insulin binding activity, autotyrosine kinase and protein-tyrosine kinase activity.  4  1.2.2 RECEPTOR TYROSINE KINASE.  The receptor tyrosine kinase region is structurally related to other tyrosine kinases including the transforming proteins encoded by oncogenes and receptors for the epidermal growth factor (EGF), platelet derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Binding of insulin to its receptor causes the autophosphorylation and activation of its B-subunit tyrosine kinase activity (Kahn and White, 1988). Site-directed mutagenesis of the receptor ATP binding site abolishes both receptor autophosphorylation and biological activity, suggesting that the kinase is critical for signal transmission (Chou et al., McClain et al., 1987). However, mutant receptors with defective tyrosine phosphorylation sites and hence reduced kinase activity have been shown to retain some biological activity (Ellis et al., 1986; Wilden et al., 1990). In view of these studies as well as others concerning anti-receptor antibodies which weakly stimulate receptor autophosphorylation, the relation between kinase activity and biological activity of the insulin receptor remains a matter of debate.  5  1.3 SIGNALLING MECHANISMS IMPLICATED IN THE MECHANISM OF INSULIN ACTION.  1.3.1 PROTEIN PHOSPHORYLATIONIDEPHOSPHORYLATION SIGNALLING SYSTEM.  Several studies suggest that the tyrosine kinase activity of the B-subunit of the insulin receptor is essential to mediate the biological effects of insulin. Deletions and truncations that result in loss of insulinstimulated kinase activity or mutations in the ATP-binding site cause loss of some of the rapid effects of insulin action (Wente and Rosen, 1990; Olefsky, 1990; Kasuga et al., 1990). Similarly, inhibition of kinase activity by monoclonal antibodies impairs insulin effects (Kasuga et al., 1990; Avruch et al., 1990). This leads to the  hypothesis that the effect of insulin is mediated through tyrosine phosphorylation of cellular substrates. One potential substrate is the insulin receptor itself. This raises the possibility that a conformational change or other change induced in the receptor by autophosphorylation, rather than the increase in tyrosine kinase activity towards exogenous substrates could be involved in the transmission of the signal. This is supported by the finding that mutations of the autophosphorylation domain alter insulin responses (Shechter et al., 1989; Saperstein et al., 1989). In addition, this may also explain why some monoclonal  6  antibodies stimulate glucose and amino acid transport with no apparent activation of the tyrosine kinase (Hawley et al., 1990; Soos et al., 1989). A number of proteins are phosphorylated on tyrosine residues in response to insulin. These cellular substrates include proteins (of unknown function) of subunit Mr 15,000, 60,000, 120,000, 185,000, or 240,000 (Kasuga et a1.,1990; Avruch et al., 1990) and also recognized enzymes including phosphatidylinositol 3-kinase ( Endemann et al., 1990) and a microtubule associated protein serine/threonine kinase with a subunit Mr 42,000 (Ray and Sturgill, 1988). There are also a number of other proteins that are phosphorylated on serine/threonine residues in response to insulin. These include the receptor itself, ATP-citrate lyase, acetyl CoA carboxylase, S6 ribosomal protein, (Avruch et al., 1990; Czech et al., 1988; Denton, 1986) an isozyme of cAMP phosphodiesterase, (Degerman et al., 1990) Raf-1 kinase (Blackshear et al., 1990; Kovacina et al., 1990) and some unidentified proteins (Avruch et a/.,1990; Denton, 1986). The functional significance of the insulin-induced phosphorylation in most of these proteins is not particularly clear, although it is often assumed that some change in function occurs. Taken together, these findings suggest a phosphorylation cascade which is initiated by stimulation of the receptor tyrosine kinase leading to the activation of one or more protein serine/threonine kinases. Although insulin treatment  7  of several cell lines results in the phosphorylation and subsequent activation of various serine/threonine kinases assayed in vitro, such as MAP kinases, S6 kinases and Raf-1 kinase, none of these have been shown to be phosphorylated by the insulin receptor tyrosine kinase. Insulin also promotes dephosphorylation of several proteins (Table 1). This is probably mediated by activation of protein phosphatases. Recently, evidence has been presented that insulin stimulates type 1 phosphatase in skeletal tissue by phosphorylating its G-subunit on a specific serine (Dent et a/., 1990). This results in enhanced association of the phosphorylase with the glycogen particle and hence activation of glycogen synthase and inactivation of phosphorylase kinase and can thus explain the effects of insulin on glycogen metabolism. Other phosphatases are probably involved in the dephosphorylation of other proteins (for example, in the activation of mitochondrial pyruvate dehydrogenase; Denton et al., 1984; Thomas and Denton, 1986) but the mechanisms by which these phosphatases are activated are unknown.  1.3.2 PHOSPHOLIPID-SIGNALLING SYSTEM.  Evidence supporting a role for phospholipid or glycophospholipid hydrolysis in insulin action is fragmentary and controversial. Whereas, some authors have reported an elevation of diacylglycerol (DAG) in some cell  8  types treated with insulin (Saltiel, 1990), others have reported no changes in cells that are major targets of the hormone (Turinsky, 1990; Augert and Exton, 1988). Conflicting reports in support of or arguing against involvement of protein kinase C (PKC) in insulin action have been also presented (Cooper et al., 1990; Blackshear et al., 1987). On the one hand, phorbol esters (the tumor-promoting activators of PKC which can substitute for DAG) can mimic some of the effects of insulin on glucose transport and phosphorylation of proteins (Farese et al., 1985; Graves and McDonald, 1985; Trevillyan et al., 1985). On the other hand, phorbol esters inhibit insulin stimulated lipogenesis and antagonize insulin action on glycogen synthesis and glycogenolysis (Van de Werve et al., 1985; Ahman et al., 1984). Further, down-regulation of PKC by prolonged exposure to phorbol-l2-myristate-l3-acetate (PMA) does not necessarily abolish or even diminish rapid responses to insulin. The dual role played by phorbol esters, both as insulinomimetic agents as well as inhibitors of insulin action is indeed intriguing. However, it may be possible to accommodate these apparently conflicting observations by invoking the involvement of distinct second messengers in the mode of insulin action. PKC activation is usually accompanied with stimulation of the hydrolysis of polyphosphoinositides, leading to the generation of inositol phosphates and DAG that contains arachidonate in the C2 position. The absence of PI turnover in response to insulin  9  and the scarcity of arachidonate in the insulin-generated DAG suggest that this DAG may indeed be different, and may arise from an alternate source.  1.3.3 GLYCOSYL PHOSPHATIDYLINOSITOL (GPI)-SIGNALLING SYSTEM.  It has been suggested that an inositol phosphate glycan (IPG) released from glycosyl phosphatidylinositol is involved in the insulin-signalling system (Saltiel, 1990). Although the insulin-sensitive GPI anchor is believed to be structurally analogous to the GPIs that anchor many cell surface proteins its chemical nature has remained elusive. In addition little is definitely known about the chemical composition of the active material released from plasma membranes by insulin action. For example, the form of inositol (myo or chiro) is debated, as is the carbohydrate composition (Saltiel, 1990; Mato et al., 1987). The release of IPG would require the action of a phospholipase, and since the activity of certain phopholipases is regulated by G-proteins, it has been speculated that an insulin-sensitive phospholipase regulated via a G-protein mechanism, may account for IPG release (Burdett et al., 1990; Davis and McDonald, 1990). Data supporting this postulate is however still fragmentary, and a final point of confusion is raised by uncertainty about whether the IPG is released at the extracellular or cytoplasmic surface of the plasma membrane (Saltiel et a/., 1989; Saltiel, 1990).  10  1.4 INSULIN PROMOTES INCREASES AND DECREASES IN SPECIFIC PROTEIN SERINE/THREONINE PHOSPHORYLATION.  Studies of proteins which change activity in response to insulin have revealed that some of these proteins exhibit net dephosphorylations (Table 1). Thus, dephosphorylation of glycogen synthase increases the activity of the enzyme leading to increased glycogen deposition (Parker et al., 1983), whereas, dephosphorylation of hormone-sensitive lipase (Stralfors et a/., 1984) and phosphorylase kinase (Sheorain et al., 1984) inhibits their activities and the respective breakdown of triglyceride and glycogen stores. Similarly, pyruvate dehydrogenase is dephosphorylated and activated in response to insulin (Martin et al., 1972), leading to increased synthesis of acetyl-CoA and fatty acids. Taken together, these and other observations demonstrate that a central role of insulin action is mediated by dephosphorylation of key regulatory enzymes in intermediary metabolism. Paradoxically, insulin action also leads to rapid increases in phosphorylation of serine/threonine residues of a family of proteins (distinct from those noted above) in target cells within minutes of exposure to the hormone. Quantitatively, the insulin-stimulated protein phosphorylations^are^more^extensive^than^the dephosphorylations described above. Several major proteins  11  within fat cells are targets of this effect (Table 1) and include ATP-citrate lyase (Alexander et al., 1979; Witters, 1981), the ribosomal protein S6 (Smith et al., 1980), acetyl-CoA carboxylase (Brownsey and Denton, 1982) and proteins so far only identified according to their subunit relative molecular weight of 22,000 and 61,000 (Belsham et al., 1980; Belsham and Denton, 1980). In addition, increased phosphorylation of the B-subunit of the insulin receptor in response to insulin has also been demonstrated (Kasuga et al., 1982; Pang et al., 1985). It is important to stress that increased phosphorylation of the insulin receptor Bsubunit occurs at serine residues as well as tyrosine.  12  TABLE 1. PROTEINS EXHIBITING CHANGES IN PHOSPHORYLATION IN FAT CELLS EXPOSED TO INSULIN.  DECREASES IN PHOSPHORYLATION ^INCREASES IN PHOSPHORYLATION  Pyruvate dehydrogenase  Acetyl-CoA carboxylase  Triglyceride lipase  ATP-citrate lyase  Glycogen phosphorylase  Insulin receptor  Phosphorylase kinase  Ribosomal protein S6  Glycogen synthase  61 kD plasma membrane protein 22 kD cytoplasmic protein  Insulin-directed phosphorylation of ATP-citrate lyase (ACL) is site-specific and is apparently mediated by a serine/threonine kinase with a subunit Mr of 52,000 (Yu et al., 1990; Klarlund et al., 1990). In general insulin and hormones that elevate cAMP levels have opposite cellular effects. Surprisingly, however, both insulin and hormones which increase cAMP levels cause the phosphorylation of ACL on the same serine residue as determined by amino acid sequencing of phosphopeptides (Pierce et al., 1982; Ramakrishna et al., 1983). One possible caveat to this system is the appearance of additional phosphopeptides  13  including phosphopeptides reported by Benjamin and coworkers (Ramakrishna and Benjamin, 1985). Although insulin enhances serine phosphorylation of ACL, no apparent change in catalytic activity of the enzyme has been detected and subsequently no regulatory role has been assigned to this covalent modification, although an argument for an alteration in subcellular distribution of ACL has been proposed. The phosphorylation of the ribosomal protein S6 has been correlated with growth and increased protein synthesis (Krieg et al., 1988; Palen and Traugh, 1987). It is believed that the phosphorylated epitope on the S6 polypeptide is located within a cleft where mRNA binds (Bommer et al., 1980; Kozma et al., 1989), a location consistent with a role in regulation of initiation. Direct evidence that S6 phosphorylation leads to altered rates of initiation in vitro however, is still missing, perhaps due to the complexity of experiments involved. Furthermore, it is becoming evident that a number of proteins associated with initiation and elongation steps of protein synthesis are also substrates for protein kinases and phosphatases (reviewed by Hershey, 1991). Both the activation and inhibition of acetyl-CoA carboxylase (ACC) mediated by insulin and adrenaline, respectively, are accompanied by modest overall increases in phosphorylation of the enzyme (Brownsey and Denton, 1982; Brownsey et al., 1977; Witters, 1981). An explanation for  14  this apparent paradox came about with the demonstration that both insulin and adrenalin brought about phosphorylation but of different sites, which could be resolved by 2-dimensional mapping of tryptic phosphopeptides. The insulin-mediated activation of ACC is accompanied by phosphorylation at a serine residue located on a tryptic peptide (designated as the I-peptide, Brownsey and Denton, 1982). Adrenalin on the other hand, induces phosphorylation of the "A-sites" which are serine residues located on the tryptic peptides designated A-peptides. The latter peptides have now been sequenced (Hardie and coworkers) and their location within the primary sequence determined. Serine residues 79, 1200 and 1215 are the principal sites phosphorylated in response to rising cell cAMP (Haystead et al., 1990; Davies et al., 1990). The greater abundance of insulin-stimulated protein phosphorylations over dephosphorylations suggests their potential importance in the mechanism of insulin action. These insulin activated phosphorylations may be due to increases in kinase activities or decreased phosphatase activity, or both. In general, effects of insulin on protein phophatases have been difficult or impossible to detect once the integrity of cells is disrupted by homogenizations. Two important exceptions have been described. Firstly, within intact mitochondria, evidence of persistent activation of pyruvate dehydrogenase phosphate phosphatase has been observed following insulin treatment (Hughes and Denton,  15  1976). Very recently, evidence for activation of glycogen synthase^phosphatase^has^also^been^demonstrated. Intriguingly, the activation of glycogen synthase phosphatase appears to be mediated by activation of an insulin-sensitive protein kinase (Dent et al., 1990). Thus, in skeletal muscle, insulin appears to activate a protein serine/threonine kinase (ISPK). ISPK phosphorylates site 1 on the glycogen targeting subunit of protein phosphatase 1 (PP1G), leading to the enhancement of glycogen synthase phosphatase activity of PPIG. The latter causes sitespecific dephosphorylation at multiple serine residues on glycogen synthase and the 8-subunit of phosphorylase kinase, activating and inactivating these two enzymes, respectively. As a result, glycogen synthesis is stimulated and glycogenolysis inhibited. Clearly, in this case the activation of a protein phosphatase (PPIG) by the ISPK explains how insulin can simultaneously stimulate the phosphorylation of some proteins and the dephosphorylation of others using a common mechanism. An important element of this regulatory network is spatial separation, of key proteins in this case by binding to or released from the glycogen particles of muscle cells.  16  1.5 INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASES.  A number of insulin stimulated protein kinases have been described by various authors (see Table 2). The insulinsensitive kinases generally exhibit the critical property that they are stable after tissue disruption under carefully controlled conditions which usually include the presence of phosphatase inhibitors. This is consistent with the role of phosphorylation as a mode of regulation of these activated kinases and has facilitated further purification. In addition, the insulin-stimulated protein-serine/threonine kinases are distinct from a number of well-characterized protein kinases classically activated by a variety of second messengers. The important characteristics of some of these kinases are now described below.  17  TABLE 2 INSULIN-SENSITIVE SERINE/THREONINE RINASES ENZYME  SUBSTRATE  REFERENCE  p65-70 S6 kinase  40S ribosomes  Novak-Hofer and Thomas (1984); Cobb, 1986.  S6 kinase,rsk  40S ribosomes, glycogen synthase, S6 peptide. lamin C, G-PP1  Erikson and Mailer, 1988; Ward and Kirschner, 1990; Lavoinne et al., 1991.  p52 Kemptide kinase  kemptide, histones, ACL  Klarlund et al., 1990.  p42 Map-2 kinase^MAP-2, MBP (identical to ERK2)  Ray and Sturgill 1988; Erickson et a/.,1990.  ERK1  MAP-2, MBP  Boulton et al., 1990.  Casein kinase II  RRREEETEEE Casein  Sommercorn et al., 1987.  p70 Raf-1  histones, ACL, syntide 2, raf-1 peptide  Kovacina et al., 1990; Blackshear et al., 1990.  Insulin receptor serine kinase  insulin receptor  Smith and Sale, 1989.  Microsomal kinase  histone V-S, kemptide  Yu et a/.,1987.  Glycogen synthase kinase 3  inhibitor-2  Yang et al., 1988.  Multifunctional protein kinase (inhibited)  glycogen synthase, ACL  Ramakrishna and Benjamin, 1988.  Insulin stimulated protein kinase. (very similar to p90 s6K )  G-PPI glycogen synthase  Lavoinne et al., 1991.  18  1.5.1 S6 KINASES.  Insulin and various mitogens markedly increase the phosphorylation of ribosomal protein S6 in a variety of cell types (Smith et al., 1980; Cobb, 1986; Erikson and Mailer, 1986; Pelech et al., 1986). Phosphorylation of S6 is catalyzed by at least two distinct mitogen activated S6 kinases which are distinguishable by size, the RSK (for ribosomal S6 kinase) family containing enzymes from 90,000 to 92,000 Mr, denoted p90 rsk , initially isolated from Xenopus laevis, (Erikson and Mailer, 1985; 1986) and 60,000 to 70,000-Mr kinase activities designated p70 s6K , purified from avian and mammalian sources (Blenis et al., 1987; Jeno et al., 1988; Price et al., 1989). Both enzymes are in turn activated by serine/threonine phosphorylations in response to mitogens, and are inactivated by protein phophatase 1 and phosphatase 2A (Ballou et al., 1988a; Price et al., 1990). The activated forms of both enzymes autophosphorylate on serine/threonine residues although this does not appear to alter kinase activity towards S6. When stimulated by mitogens, neither of the two enzymes contains phosphotyrosine. This suggests that serine/threonine kinases in the upstream pathways may be responsible for the activation of these S6 kinases. Intriguingly, phosphotyrosine has been detected in p90 rsk which was activated by the Rous sarcoma virus protein pp60 src (Sweet et al., 1990). The significance of this tyrosine  19  phosphorylation is unclear because of the absence of phosphotyrosine in p90 rsk stimulated with other mitogens. The structure of the p90 rsk enzymes is highly conserved in other species; cDNA clones have been used to identify similar enzymes expressed in chicken, mice and humans (Alcorta et al., 1989). These enzymes contain a unique two kinase catalytic domain motif. The N-terminal half of the molecule is related to protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and cGMP-dependent protein kinase whereas, the C-terminal half is related to the catalytic subunit of phosphorylase b kinase (Jones et al., 1988). While the functional significance of this motif remains unknown, it is conceivable that both domains are vital elements during the activation and function of these kinases. p70S6K b y contrast contains a single catalytic domain  57% identical to the N-terminus of p90 rsk . The C-terminal region of this enzyme shares some sequence homology with the S6 substrate itself and is proposed to present a pseudosubstrate site (Banerjee et al., 1990) which may prevent substrate-enzyme interaction and/or ATP binding. It is also believed that phosphorylation of this region of p7 0S6K by yet an unidentified mitogen-activated kinase  relieves the constrains of this inhibitory domain, leading to activation of the enzyme (Price et al., 1991). Evidence for the regulation of p90 rsk is also still very scanty. Blenis and coworkers (Chung et al., 1991), have  20  recently reported the presence of a kinase activity in Swiss 3T3 cells able to phosphorylate an inactive rsk-encoded protein. It is believed that MAP kinases may be involved in the upstream regulation of the p90 rsk kinases. Both purified S6 kinase enzymes phosphorylate 40S ribosomal subunits to high stoichiometries (4-5 mol P/mol protein), with high specific activities of hundreds of nmol/min/mg (Kozma et al., 1990; Banerjee et al., 1990; Erikson and Mailer, 1985, 1986, 1988). Phosphopeptide maps of S6 phosphorylated in vivo in response to mitogens and in  vitro by the purified enzymes are identical, implying that both kinases function in vivo as S6 kinases. p70 s6K is probably more specific for 40S ribosomes as a substrate whereas, p9O rsk phosphorylates several other substrates in  vitro with a consensus sequence of R-X-X-Ser (Erikson and Maller, 1988). Thus, the specificity of p90 rsk for S6 is not absolute, implying a possible multifunctional, physiological role for this enzyme. In terms of time-course, the activation of the S6 kinases is biphasic or even multiphasic (Sweet et al., 1990; Ahn et al., 1990; Susa et al., 1989). p90 rsk is maximally activated within the first few minutes of mitogenic challenge and thus, activation is often transient. In contrast, the activation of p7 0S6K although initiated rapidly, usually reaches a maximum in 30-60 minutes and the activation is more sustained. According to some authors, the maintained activation of p70 s6K may require the activation  21  of protein kinase C (Susa et al., 1989), but this is debated. Two partially purified EGF-stimulated MBP kinase activities from Swiss 3T3 cells identified as E3 and E4 with native molecular weights of approximately 30 and 50 kDa respectively have been shown to stimulate an S6 peptide kinase activity (B1) in vitro that behaved as an 110 kDa protein on Superose (Ahn and Krebs, 1990). Finally, using recombinant RSK peptide as an in vitro substrate, two chromatographically distinct, serum-activated RSK kinases, designated RSKI (p44) and RSK II (p42) were identified in Swiss 3T3 cells (Chung et al., 1991b). Both of these kinases are tyrosine phosphorylated and are immunological related to a meiosis-activated protein kinase from maturing sea star (p44 mP k ). Furthermore, these protein kinases phosphorylated and partially activated the ribosomal p90 rsk . The numbers of and the precise inter-relationship of all these kinases to one another is still not entirely clear although the relationships are emerging rapidly as the cloning and sequencing of MAP kinase isoforms progresses.  1.5.2 p42 and p44 MAP (MITOGEN-ACTIVATED PROTEIN) KINASES.  Studies by Sato and coworkers (1985) demonstrated that insulin and other mitogens stimulated the phosphorylation of cytoskeletal-associated proteins. Later studies using microtubule-associated protein-2 (MAP-2) showed the presence  22  of kinase activities from cells stimulated by various mitogenic agents (Ray and Sturgill, 1987; Hoshi et al., 1988). The acronym MAP initially taken from the preferred in vitro substrate microtubule-associated protein-2, now stands for mitogen-activated protein kinase, consistent with the relatively promiscous activation induced by diverse mitogenic agents (Rossomando et al., 1989). At least two closely related murine MAP kinases are known to exist and they exhibit apparent subunit Mr of 42,000 (p42 ma P k ) and 44,000 (p44 maPk ). Activation of these MAP kinases is rapid and transient reaching a maximum within the first 10 minutes of stimulation (Ahn et al., 1990; Ray and Sturgill, 1987; Ahn and Krebs, 1990) and subsequently becoming inactive within the next 60 minutes. Whereas, microtubule associated protein-2 and myelin basic protein are preferred substrates for the MAP kinase, casein, histones, the S6 peptide, and 40S ribosomal subunits are not appreciably phosphorylated (Ray and Sturgill, 1988; Erickson et al., 1990). M-phase arrested Xenopus eggs contain tyrosine phosphorylated 40-45 kD proteins (Cooper, 1989). Pelech and coworkers have purified to homogeneity a 44 kDa MBP-kinase (designated p44 mP k for meiosis-activated protein kinase) from maturing sea star (Sanghera et al., 1990). p44 mP k is immunologically related to murine p42 ma P k and p44 ma P k . However based on molecular weight and copurification during phenyl-Superose, polylysine and MonoQ chromatography p44mPk  23  and p44 ma P k seem even more closely related (Rossomando et al., 1991). In addition to those studies of protein kinases above, an additional molecular cloning approach by Cobb and coworkers has led to the recognition of four or more protein serine/threonine kinases (initially using a rat brain cDNA library) which are highly related and termed "ERKs" for extracellular-signal regulated kinases (Boulton et al., 1991a; Boulton and Cobb, 1991). An insulin-stimulated microtubule-associated protein-2 kinase sequenced by Boulton and coworkers termed ERK1 (Boulton et al., 1990) is also closely related to p42 ma l* and p44 maP k . ERK1 has a subunit molecular size of 43 kDa as judged by SDS-PAGE (Boulton et al., 1991b). In addition, it is inactivated completely by phosphatase 2A and substantially by CD45, suggesting regulation through serine/threonine and tyrosine phosphorylations.  1.5.2.1 ACTIVATION OF 90 kDa S6 KINASE BY MAP KINASE.  Although S6 kinases are activated by serine/threonine phosphorylations,^the kinases responsible for this activation have not been completely established. Most intriguing is how signaling mechanisms from the insulin receptor tyrosine kinase are linked with protein serine/threonine kinases. A possible candidate for this link is the family of MAP kinases. When phosphatase-inactivated  24 p 9 o r sk from Xenopus oocytes was incubated with insulin-  activated MAP kinase from 3T3-L1 cells, partial reactivation and phosphorylation of the enzyme was observed (Sturgill et al., 1988). Phosphorylation was found on threonine residues, at sites distinct from the p9O rsk autophosphorylation site, implying that these enzymes constitute a single step of a protein serine/threonine kinase cascade. Although the MAP kinases do become insulin activated with parallel tyrosine phosphorylation a direct link of MAP kinase with the receptor tyrosine kinase has, however, not been proved. Thus, purified MAP kinase has not been successfully phosphorylated by preparations of insulin receptors.  25  1.6 THESIS INVESTIGATION.  The general hypothesis is that the actions of insulin on cell metabolism and function are mediated by mechanism involving reversible phosphorylation of proteins. It has been recognized that insulin leads to increases in fatty acid biosynthesis through activation of several enzymes including ACC (which demonstrated insulin-induced phosphorylation). Insulin activates protein-serine/threonine kinases in fat cells and this thesis describes my attempt to purify and characterize such insulin-activated fat cell protein serine/threonine kinases. As model target substrates for the kinases to be studied, myelin basic protein and ACC have been employed. Initial studies had revealed the presence of a number of insulin-sensitive serine/threonine kinases in extracts from rat adipose tissue. Progress was severely hampered because the kinase activities proved to be very labile even after employment of a number of standard conditions for the stable storage of proteins. This necessitated devising means of stabilizing the kinase activities and particularly important was rapid initial partial purification and use of new protein phosphatase inhibitors. In addition to the protein purification, parallel characterization employing several different antisera including antibodies, which recognize phosphotyrosine and antiserum raised against purified p44mPk  26  (a meiosis-activated protein kinase from sea star), has provided an important insight into the complexity of and success in purification of the specific proteinserine/threonine kinases of interest. The development of this improved purification technique will provide a strong basis for the future studies, which will enable further molecular characterization of the insulin-stimulated kinase activity.  27  MATERIALS AND METHODS. 1.1 MATERIALS.  Male Wistar rats (140-160 g) supplied by the University of British Columbia (UBC), Animal Care Unit were housed in the Department of Biochemistry 2-3 days prior to use. The animals maintained on a 12 hour light-dark cycle were allowed free access to water and Purina rat chow until the time of killing, usually between 8am and 9am. Most laboratory chemicals and solvents were obtained from BDH Chemicals Canada Ltd. The biochemicals including the protease inhibitors (pepstatin A, leupeptin and benzamidine) as well as bovine myelin basic protein (MBP), synthetic inhibitor of cAMP-dependent protein kinase and mouse monoclonal antiphosphotyrosine antibody (PT-66) were purchased from Sigma Chemical Company (St. Louis, MO.,USA). Microcystin LR was bought from Calbiochem. Human MBP was a generous gift from Frank Nezil and Dr. Myer Bloom of the Physics department at UBC. The antibodies R1, R2, GEGA and anti-p44 mP k were generously supplied by Dr. S.L. Pelech at the Biomedical Research Centre, UBC. Initial trial supplies of antiphosphotyrosine antibodies and antiphosphotyrosineagarose were kindly supplied by Alice Mui and Dr. Jerry Krystal (Terry Fox lab., BC Cancer Control Agency, Vancouver, B.C.).  28  All radiochemicals including [gamma- 32 P]ATP as well as ACS scintillation fluid and the rainbow molecular weight markers for polyacrylamide gel electrophoresis (PAGE) were purchased from Amersham International (Oakville, Ontario, Canada). Reagents for PAGE were bought from Bio-Rad Laboratories (Canada) Ltd, Mississauga, Ontario.  1.2 METHODS.  1.2.1 TISSUE INCUBATION AND PREPARATION OF EXTRACTS.  Epididymal and perirenal fat pads were freshly isolated from normally-fed male Wistar rats and immediately incubated at 37°C in a bicarbonate buffered medium which had been gassed with 02:CO2 (19:1) for 20 min prior to warming to 37°C. This was followed by an additional 10 min incubation in a fresh medium of the same composition with or without addition of insulin (0.5 gg/ml). At the end of the incubation period, pads were removed from the medium, lightly and rapidly blotted on Whatman filter paper. The tissue was then disrupted at 0°C with a Polytron PT-15-35 tissue homogenizer (setting 6, 3s) into buffer, pH 7.2, containing 250 mM- sucrose, 50 mM-Mops, 40 mM-p-nitrophenyl phosphate (pNPP), 5 mM-EDTA, 2 mM-EGTA, 1 mM-sodium orthovanadate, 1 mM-DTT, 1AM-8-methylaspartic acid, 100 nMmicrocystin LR, 1 mM-PMSF, 2.5 mM-benzamidine and 2 gg/ml  29  each of pepstatin A and leupeptin. The extraction used 4 ml buffer per gram fresh weight of tissue. A soluble fraction was prepared from the adipose tissue extracts by centrifugation. This consisted of an initial centrifugation at 15, 000 xg for 10 min (SS-34/Sorval). The fat-free infranatant was further centrifuged at 360,000 xg av for an additional 10 min in a benchtop ultracentrifuge (TLA 100.1/Beckman) or alternatively using a conventional ultracentrifuge at 215,000 xg av for 1 hour (Ti 70/Beckman). The high speed supernatant (HSS) fraction obtained was either used directly or was subsequently fractionated using ammonium sulphate or MonoQ ion exchange chromatography as described below. 1.2.2 AMMONIUM SULPHATE PRECIPITATION.  Saturated ammonium sulphate solution (761 g/1; Scopes, 1987) was adjusted to pH 7.5 at 4°C with Tris buffer. An equal volume of this solution was added slowly with stirring to an equal volume of the HSS at 4°C. Stirring was continued for 1 h. This was followed by centrifugation at 27,000 xg for 30 min (SS-34/Sorval). The pellets were either resuspended and tested directly or stored at -70°C until further chromatography usually carried out the following day. For resuspension, ammonium sulphate pellets were dissolved at 0°C in a minimum volume of appropriate buffer with gentle mixing and after incubation at 0°C for 20-30  30  minutes were clarified by centrifugation (5 min, 10,000 xg, Eppendorf cetrifuge).  1.2.3 ASSAY OF PROTEIN KINASE ACTIVITY.  Protein kinase assays were carried out in a final volume of 50 gl and contained 0.1 mg/ml MBP (or other protein substrate as indicated), 5 mM-MgC12, 40 µM-[gamma- 32 1:]ATP (1000 dpm/pmole), 0.6 gM of the peptide inhibitor of cAMPdependent protein kinase (3750 U/ml), 50 mM-Mops pH 7.5, 2 mM-EGTA, 1 mM-DTT and 1 gM-B-methylaspartic acid. All reaction preincubations were performed at 0°C. After brief incubation (1-2 min at 30°C), the kinase reactions were initiated upon addition of [gamma- 32 P]ATP and proceeded for 10 min at 30°C. The assay reactions were terminated by spotting 30 gl aliquots of reaction mixture onto Whatman P81 phosphocellulose paper (Glass et al., 1978). The filter papers were washed 10 times with gentle shaking on an ice bath (over a total of approximately 2 h) in 0.85% phosphoric acid. Washed papers were then transferred into 20-m1 glass scintillation vials containing 10 ml distilled water and [ 32 P] incorporation quantified by Cerenkov counting. Alternatively, some reactions were terminated by addition of 500 gl of pre-chilled acetone (-20°C). After incubation at -20°C for 2 h, precipitated proteins were recovered by centrifugation (5 min, Eppendorf) and then subjected to SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The gels were  31  stained with Coomassie blue, destained and finally washed with acetic acid (7% w/v) containing glycerol (4%) to stabilize and hence minimize cracking. The destained gels were air-dried and subjected to autoradiography using Kodak XAR film at -70°C in cassettes (Rigid form, Brooklyn, NY) containing Dupont Hi plus Cronex intensifier screens. This was followed by densitometric scanning of the autoradiograms using a Biorad video densitometer model 620.  1.2.4 SDS- POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE).  Proteins were dissolved in 30-50 gl sample buffer containing 62.5 mM-Tris pH 6.8, 5% SDS (w/v), 20% sucrose (w/v), 0.2 mg/ml bromophenol blue, 5% 2-mercaptoethanol (v/v) and separated on 1.5 mm thick slab gels by the discontinuous buffer system of Laemmli (1970). Gels were usually standard 7.5 (w/v) or gradient 7.5-15% (w/v) with respect to acrylamide concentration and were run under constant current 20 mA per gel. The gels were then fixed in solution containing trichloroacetic acid (20%, w/v) and methanol (40%, w/v) for 10 min, stained with Coomassie blueR250 in acetic acid (7% w/v), methanol (45% w/v) for 30 minutes, destained (acetic acid:methanol, mixtures as above) and air-dried between clear membrane sheets (Bio-Rad), which were clipped to glass plates.  32  1.2.5. SILVER STAINING OF GELS.  Gels were soaked overnight in methanol (50% v/v) and then washed in Milli-Q water with two changes over 30 min. The silver staining method was essentially adopted from Rabilloud  et al.,  (1988). The gels were soaked for 1 min in  a sensitizing solution containing sodium dithionite (0.25 g/l) and rinsed in Milli-Q water for another minute. The gels were then incubated in a staining solution containing silver nitrate (0.2% w/v, in 1 mM formaldehyde) for 30 min with shaking. After a final rinse in Milli-Q water the stain was developed in a solution containing potassium carbonate (4% w/v) in 6 mM formaldehyde and 20 M sodium thiosulphate. After 10 min, the reaction was stopped by addition of glacial acetic acid (3.5 ml/100 ml) directly to the developer.  1.2.6 AUTORADIOGRAPHY.  Kodak X-ray film (X-Omat XAR-5) was preflashed and exposed at -70°C to polyacrylamide gels or TLC plates in cassettes with intensifying screens (Dupont Cronex Hi-Plus). After exposure, the film was developed in an automatic developer (Kodak M35A X-OMAT Processor).  33  1.2.7 SCANNING DENSITOMETRY.  Scanning densitometry of autoradiograms was performed using a Biorad model 620 video densitometer and data was analyzed using the Biorad 1-D Analyst (version 2.0) computer software package.  1.2.8 CHROMATOGRAPHY.  In general, to facilitate rapid chromatographic separation, a Fast Protein Liquid Chromatography (FPLC) system (Pharmacia) was used. The system employed an automatic sample injection valve (MV-7), dual pumps for programmable isocratic or gradient elution and a detector to allow determination of the UV absorption of column effluent.  1.2.8.1 DEAE-CELLULOSE ION EXCHANGE CHROMATOGRAPHY.  High speed supernatant (HSS) fractions from rat adipose tissue stored at -70°C as ammonium sulphate pellets were allowed to thaw and resuspended in 3 ml of buffer A (50 mMTris/HC1 pH 7.5 at 4°C, 1 gM B-methylaspartic acid, 2 mMEGTA, 2.5 mM-benzamidine, 2  µg/ml  each of pepstatin A and  leupeptin, and 2 mM-DTT). The resuspended pellet was applied to an Econo-Pac 10 DG desalting column (Bio-Rad) and the desalted sample collected in a 5 ml-volume. The sample was applied to a DEAE-cellulose column (2.5 x 6 cm) at a flow  34  rate of 10 ml/h. After washing the column at 20 ml/h with 20 ml of buffer A, the column was eluted at 10 ml/h with buffer A containing 0.5 M NaCl. Ten 2-ml fractions were collected. Fractions containing kinase activity were pooled, concentrated using a centricon 10 microconcentrator (Amicon) and stored at -70°C.  1.2.8.2 PHENYL-SEPHAROSE CHROMATOGRAPHY.  Ammonium sulfate was added to the DEAE-cellulose sample up to 0.8 M and the sample applied to a phenyl-Sepharose column (2.5 x 6 cm) previously equilibrated in buffer A containing 0.8 M ammonium sulphate. The column was washed with 3-5 column volumes of the loading buffer and then eluted with three sequential steps: firstly with buffer A, secondly with buffer A containing 50% ethylene glycol and finally with 2% (w/v) Nonidet P40 in buffer A. In some cases indicated in the results section, a different elution procedure was used in which the column was eluted only with 3% Brij 35.  1.2.8.3 MONOO ION EXCHANGE CHROMATOGRAPHY.  To ensure removal of all particulate material, the desalted ammonium sulphate pellet was filtered through a Syrfil 25 mm 0.22 gm filter (Amicon) and applied to a MonoQ anion exchange column (HR 5/5) in buffer A. To ensure the  35  removal of unbound protein, the column was washed with 5 column volumes of buffer A at a flow rate of 1 ml/min. The column was developed by a linear gradient of 0-0.5 M NaC1 also using buffer A and at the same flow rate. Forty 1-ml fractions were collected assayed for kinase activity and stored at at -20°C.  1.2.8.4 AFFINITY CHROMATOGRAPHY USING MBP-AFFIGEL COLUMN.  1.2.8.4.1 COUPLING OF MBP TO AFFIGEL 10.  One milliliter of Affigel 10 (Bio-Rad) was washed sequentially with 3 ml each of isopropanol and cold deionized water. To the washed gel was added 5 mg of bovine MBP dissolved in 0.5 ml 50 mM Mops pH 7.5. The protein/gel mixture was incubated with shaking for 1 h at room temperature and following addition of 0.1 ml ethanolamine.HC1, pH 8.0, the incubation was continued for another 1 h in order to block unoccupied sites of the Affigel matrix. Blocking is particularly important when the gel is to be used immediately. After blocking with ethanolamine, the gel was transferred to a column and the protein solution, together with 3 subsequent washes with solution containing 7 M urea/1 M NaC1 each were collected. The protein concentration in the wash fraction was determined using the Bradford assay and on the basis of this the coupling efficiency was estimated to be 98%.  36  1.2.8.4.2 MBP-COLUMN CHROMATOGRAPHY.  A simple two-step batchwise elution procedure was attempted to investigate the possible binding of protein kinase activity to MBP-Affigel. One milliliter of protein fraction containing MBP-kinase activity which had been partially purified by DEAE-anion exchange chromatography was applied to the MBP-column. After washing with 3 ml of buffer B (buffer B = buffer A with 50 mM Mops instead of 50 mM Tris), the column was eluted with 0.5 ml buffer B containing 0.5 M NaCl.  1.2.9 WESTERN BLOTTING ANALYSIS.  1.2.9.1 SDS-PAGE AND TRANSFER OF PROTEINS.  One hundred microliter samples from each of the fractions obtained following chromatography with MonoQ, phenyl-Sepharose or other techniques as indicated were precipitated with 1 ml of cold acetone (-20°C). The samples were incubated at -20°C for 2 h and centrifuged for 5 min (Eppendorf). After removing the acetone by aspiration, the pellets were allowed to dry in order to remove the residual acetone. The pellets were dissolved in 30-50 gl of SDSsample buffer by heating at 95°C for 5-10 min. The proteins  37  were separated on 7.5-15% gradient gels using SDS-PAGE as described before. The separated proteins were electrophoretically transferred to Immobilon PVDF membrane (Millipore) in 20 mM-Tris/acetate pH 7.4, 2 mM-EDTA, 0.01% SDS (w/v) at 400 mA for 90 min. The blots were washed briefly in KBS (8 g/1 NaC1, 0.2 g/1 monobasic, potassium hydrogen phosphate, 1.93 g dibasic, sodium hydrogen phosphate, heptahydrate, 0.02% sodium azide (w/v), 0.2 g KC1 and 0.5 ml/L Tween 20), and then stained in 0.2% Ponceau S / 3% TCA / 3% sulfosalicylic acid. The blots were destained in KBS and blocked in KBS containing 0.5% Tween-20 for 1 h at room temperature or overnight at 4°C.  1.2.9.2 ANTIBODY BINDING.  The blots were removed from the blocking solution and washed twice for 5 min each in KBS. Blots were probed with the indicated antibodies for 2 h at room temperature. The antibodies used included: affinity purified rabbit polyclonal antibodies raised against purified sea star p44 mP k (1:1000) (Sanghera et al., 1991), "R2," and "R1" rabbit antisera raised against the carboxy terminal region (with the sequence PFTFDMELDDLPKERLKELIFQETARFQPGAPEAP) and kinase subdomain III (with the sequence PFEHQTYCQRTLREIQILLGFRHENVIGIRDILRAP), respectively, of ERK1 (1:1000), rabbit antiserum developed against a synthetic  38  peptide^(GEGA)^with the^sequence^GLAYIGEGAYGMVAC corresponding to protein subdomain I of sea star p44 mP k (1:1000) or anti-phosphotyrosine (PT-66, Sigma) (1:2000) for 2 h at room temperature. After washing with 5 changes of KBS over 25 min, the blots were incubated with a secondary antibody conjugated to alkaline phosphatase for 1 h. The secondary antibody was raised in goat against rabbit IgG. After washing 4 times in KBS as above and once in distilled water, the membranes were incubated in substrate buffer (100 mM-NaCl, 5 mM-MgC12, 100 mM-Tris/HC1 pH 9.5), and finally the blots were developed with bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate. 1.2.10 IMMUNOPRECIPITATION.  Two different kinds of mouse monoclonal antibodies raised against phosphotyrosine, PT-AM (a gift from Alice Mui, Terry Fox lab, Vancouver) and PT-66 (Sigma) were used in an attempt to immunoprecipitate the MBP-kinase activity. 200 Al of MBP-kinase activity partially purified by MonoQ ion exchange chromatography was incubated with 5-20 Al of the antibody overnight at 4°C. The incubation was performed in a buffer which contained 100 AM sodium orthovanadate. 1050 Al recombinant protein G (Zymed) was then added and the incubation continued for 30 min. After the supernatant was removed, the protein G pellet was washed 3 times with 1 ml 20 mM triethanolamine buffer pH 7.5, containing 0.5% (w/v) Nonidet P40 and 0.5M NaCl. After a final rinse in distilled  39  water, 50 Al of SDS sample buffer was added and the sample heated at 95°C for 10 min. The proteins were separated using SDS-PAGE followed by electrophoretic transfer to immobilon membrane. The blot was probed with R2 and anti-p44 mP k antibodies. 1.2.11 PROTEIN ASSAYS.  Protein was assayed by the method of Bradford (1976). Two hundred microliter samples containing 1-20 Ag of protein were mixed with 1 ml of Bradford reagent and the colour allowed to develop at room temperature. After 15 min the absorbance was measured at 595nm. Protein concentration was estimated from a standard calibration curve, linear only up to 15 gg gamma-globulin, and which was prepared on each separate occassion. The values shown are the mean of duplicate samples.  40  RESULTS  1.0 INSTABILITY OF PROTEIN KINASE ACTIVITY IN EXTRACTS OF RAT FAT PAD TISSUE.  Previous experiments showing the presence of insulin stimulated kinases were carried out using freshly-prepared fractions from rat adipose tissue. In such experiments it had been possible to carry out rapid MonoQ chromatography immediately after preparation of high-speed supernatant fractions and then detect peaks of insulin-stimulated protein kinase activity directed against different phosphoacceptor substrates. In these earlier experiments, all kinase assays were carried out on the same day as the tissue extractions. Further developments in the rapid purification and characterization were retarded by the loss of protein kinase activity. The insulin-activated protein serine/threonine kinases proved to be particularly labile, leading to loss of apparent insulin effect with storage of fractions either at 4°C, 0°C, -20°C or -70°C as shown in Table 3. The decay in protein kinase activity occurred despite inclusion of commonly used protein phosphatase inhibitors (EGTA, EDTA) and proteolytic enzyme inhibitors. Other common phosphatase inhibitors including sodium fluoride and B-glycerolphosphate appeared to directly inhibit adipose tissue kinase activities and were therefore unsuitable. More specifically, extraction buffers identical  43.  to those used for the preservation of ribosomal S6 kinases (used by Novak-Hofer and Thomas, 1984) also proved unsuccessful in stabilization. The immediate concern was therefore, to devise means of preserving these insulin sensitive protein serine/threonine kinase activities of interest.  42  TABLE 3. THE LOSS OF INSULIN-STIMULATED KINASE ACTIVITY UNDER DIFFERENT STORAGE CONDITIONS.  STORAGE CONDITION^  % LOSS OF ACTIVITY  50% GLYCEROL, -70°C, 2 DAYS^  92  4°C, 10 DAYS*^  89  CONCENTRATED, -70°C, 6 DAYS^  67  High speed supernatant fractions from rat adipose tissue were fractionated with ammonium sulphate (50%), desalted and applied on a MonoQ column. The column was developed with a linear NaC1 0-0.5 M gradient as described in Methods section. Fractions were assayed for myelin basic protein phosphotransferase activity before and after storage under different conditions. *Similar loss is also found under these conditions within 12 days at 4°C.  43  2.0 STABILIZATION OF INSULIN-ACTIVATED KINASES.  2.1 EFFECT OF PHOSPHATASE INHIBITORS.  Within the last 3-4 years, it has become clear that the activation of a number of mitogen-activated kinases can be reversed by treatment with purified protein phosphatases (Mailer, 1987; Ballou et al., 1988b; Anderson et al., 1990; Sanghera et al., 1991). These findings are consistent with a role of phosphorylation in the regulation of these kinases and consequently when these kinases were purified, buffers containing phosphatase inhibitors were used. Thus, if the insulin-activated kinases of adipose tissue are also regulated through protein phosphorylation, then the use of buffers containing protein phosphatase inhibitors would be expected to have stabilizing effects. As noted above, initial studies had indeed taken account of this possibility but the precautions successful in other studies were not adequate in this case. Attempts were therefore made to investigate which of the phosphatase inhibitors were most effective in preserving the kinase activity in adipose tissue extracts. This was carried out in the following two experiments.  44  2.1.1 ESTIMATION OF PHOSPHATASE ACTIVITY IN CRUDE EXTRACTS OF FAT PAD HIGH SPEED SUPERNATANTS.  Fat pads treated with insulin were homogenized in a buffer containing Mops (50 mM), sucrose (250 mM), EGTA (2 mM), EDTA (4 mM), DTT (2 mM) and the protease inhibitors pepstatin, leupeptin and PMSF. Variations of the above buffer were made by further additions of either 1 mM sodium orthovanadate, 1 gM B-methylaspartic acid, or 10 mM Bglycerolphosphate. The breakdown of p-nitrophenylphosphate (pNPP) to p-nitrophenol with subsequent measurement at 405 nm was used as an indicator of phosphatase activity. Unfortunately, this assay was not sensitive enough to detect changes in phosphatase activity in the presence of different inhibitors over time-course of less than 30-60 min. However, storage (even at 0°C) of supernatant fractions led to marked hydrolysis of pNPP over a period of several hours. For the purposes of protein purification, therefore, the endogenous protein phosphatases still displayed significant activity.  45  2.1.2 EFFECTS OF MICROCYSTIN ON THE PHOSPHORYLATION OF MYELIN BASIC PROTEIN BY PROTEIN KINASES PRESENT IN RAT ADIPOSE TISSUE.  It has been most fortuitous that two extremely potent inhibitors of protein phosphatases 1 and 2A (major fat cell protein phophatases) have recently been discovered. These are okadaic acid and microcystin. The effects of microcystin, a potent inhibitor of type I (PP1) and type 2A (PP2A) protein phosphatases (Honkanen et al., 1990) were investigated in an experiment whereby the phosphorylation of MBP by serine/threonine kinases present in fresh high-speed supernatant fraction of rat adipose tissue was carried out. As shown in Figure la, the phosphorylation of MBP markedly increased in a microcystin dose-dependent manner. Since microcystin inhibits protein phosphatase 1 and 2A, this observation implies that PP1 and PP2A may be the dominant protein phosphatases acting on phosphoproteins in rat adipose tissue. The extent of phosphorylation was quantitated by densitometric scanning and the results are illustrated in figure lb. Microcystin increased the phosphorylation of MBP by a factor of 2.5. The maximum phosphorylation was achieved at a microcystin concentration of 100 nM. In view of the above findings, microcystin, B-methylaspartic acid, B-glycerolphosphate, pNPP and orthovanadate were subsequently included in the extraction buffer.  46  FIGURE la. EFFECT OF MICROCYSTIN ON PHOSPHORYLATION OF MBP BY PROTEIN KINASES PRESENT IN HIGH SPEED SUPERNATANT FRACTIONS.  Rat fat pads were incubated with insulin (10 mU/m1 for 10 min) and homogenized in the absence or presence of the phosphatase inhibitor microcystin at the indicated concentrations. Kinase assays were initiated by the addition of [gamma- 32 P]ATP and allowed to proceed for 10 minutes. The phosphoproteins were separated by SDS-PAGE followed by autoradiography. The autoradiograms shown, illustrate the incorporation of 32 P into added MBP. The arrows indicate the two major bands of MBP which are phosphorylated.  47  FIGURE lb. EFFECT OF MICROCYSTIN ON THE PHOSPHORYLATION OF MBP.  Rat fat pads were incubated with insulin (10 mU/ml for 10 min) and homogenized in the absence or presence of the phosphatase inhibitor microcystin at various concentrations as shown in Fig. la. Kinase assays were initiated by the addition of [gamma- 32 P]ATP and allowed to proceed for 10 min. The phosphoproteins were separated by SDS-PAGE which was followed by autoradiography. J2 P-incorporation into MBP was assessed by densitometric scanning of the autoradiograms as illustrated below: series 1 and 3 correspond to 32 Pincorporation into lower and upper bands of MBP respectively, series 2 corresponds to average values of series 1 and 3. In each case, the values are the average of triplicate measurements in one experiment.  120  Phosphorylation (% maximum)  100 80 60 40 20 0  0 nM  ^  10 nM^100 nM  ^  1000 nM  Microcystin Concentration MI Series 1  Series 2^P i Series 3  48  2.2 AMMONIUM SULPHATE PRECIPITATION.  To address further, the question of preserving the kinase activity, a rapid initial purification step employing ammonium sulphate precipitation was considered. This technique offers the benefits of speed, large volume capability and a rather generalized stabilization of proteins (Scopes, 1987). The high salt concentration inhibits enzyme action and therefore, protects against possible proteolysis and/or phosphatase action. The actual percentage saturation of ammonium sulphate required to precipitate the kinase activity, was determined in a trial fractionation with three different cuts. As shown in Table 4, the majority of the protein kinase activity precipitated with ammonium sulphate between 20-45%. The combination of the use of phosphatase inhibitors and rapid ammonium sulphate precipitation was very effective in recovering and stabilizing protein kinase activity. Not only were the activities enriched but they could be stored for weeks at 70°C with apparently very little loss in activity, as will be demonstrated by the subsequent studies aimed at further purification.  49  TABLE 4. PRECIPITATION OF MYELIN BASIC PROTEIN-KINASE.  PERCENT^PERCENT ^ SATURATION^ENZYME PURIFICATION ^ RANGE^PRECIPITATED FACTOR  0-20  4  <1.0  20-45  85  2.7  45-65  5  <1.0  65 supernatant  6  <1.0  High speed supernatant fractions were prepared following homogenization of rat adipose tissue which had been incubated with insulin (10 mU/ml for 10 min) and then were fractionated with 0-20, 20-45 and 45-65 % ammonium sulfate. The resulting protein fractions precipitated were redissolved and then assayed for kinase activity able to phosphorylate myelin basic protein.  50  3.0^PURIFICATION^OF^INSULIN-ACTIVATED^PROTEIN SERINE/THREONINE KINASES FROM RAT ADIPOSE TISSUE.  In the long term, a high priority is to obtain an adipocyte protein kinase preparation able to phosphorylate the insulin-directed site (I-site) on acetyl-CoA carboxylase (Brownsey and Denton, 1982; 1987), since this has not been described before. Furthermore, the relationship of this activity (or activities) with respect to those involved in phosphorylation of other insulin targets especially ATPcitrate lyase is also of major interest. The insulindirected phosphorylation of acetyl-CoA carboxylase occurs only at one or two of eight possible phosphorylation sites and therefore analysis requires very lengthy phosphopeptide separation and quantitation. For convenience, we therefore chose to use myelin basic protein.(MBP) as a model substrate for protein kinase assays. The choice of this substrate was justified because (a) MBP (and MAP-2) appears to be an excellent substrate for mitogen-stimulated kinases in a number of studies. (b) a MBP kinase from sea-star oocytes has been shown to phophorylate acetyl-CoA carboxylase on the insulindirected site (Pelech et al., 1991). Initially, our approach was to carry out the preliminary isolation and stabilization of insulin-stimulated kinases as described above (employing preparation of high-speed  51  supernatants followed by ammonium sulfate precipitation). Following this, a range of different columns were employed to test for their effectiveness in purifying kinases of interest. The objective was therefore to be able to design a purification protocol first, based on sequencial column chromatography. Table 5 summarizes the various resins which were tested in an attempt to purify the insulin-activated kinases. A number of these columns had pitfalls and although not likely to be used in a final purification scheme, may nevertheless offer some important insights about the kinases. Chromatography on resins which provided separation and reasonable recovery of the activities of the insulinactivated kinases is now described below.  52  TABLE 5. THE VARIOUS CHROMATOGRAPHIC MATRICES USED IN ATTEMPTS TO PURIFY INSULIN-ACTIVATED PROTEIN SERINE/THREONINE KINASES.  MATRIX  COMMENT  DEAE-CELLULOSE  Useful for rapid early purification.  MONOQ  Excellent resolving power and recovery of kinase activity.  HYDROXYLAPATITE  Good matrix but very slow flow rates.  TYROSINE-AGAROSE  No distinct separation, enzyme activity smeared in many fractions.  Fe-CHELATING^Low pH required to bind enzyme was (IDA-SEPHAROSE)^denaturing. PHOSPHOCELLULOSE^Very weak interaction with the matrix. PHENYL-SEPHAROSE^Offered high recovery of enzyme activity. MBP-AFFINITY^Not reproducible. SUPEROSE 6^Limited by the sample volume and SEPHACRYL S-200^enzyme diluted, but useful at the SEPHADEX G-100^qualitative level. POLYLYSINE-AGAROSE^Resolved two broad peaks of kinase activity. HEPARIN-AGAROSE^Enzyme did not bind but many contaminating proteins did.  53  3.1 APPLICATION OF VARIOUS CHROMATOGRAPHIC TECHNIOUES FOR PURIFICATION OF INSULIN-ACTIVATED SERINE/THREONINE KINASES FROM RAT ADIPOSE TISSUE.  3.1.1 MONOO CHROMATOGRAPHY OF INSULIN-ACTIVATED SERINE/THREONINE KINASES FROM RAT ADIPOSE TISSUE.  High speed supernatant fractions from rat adipose tissue treated with or without insulin were subjected to 50% ammonium sulphate precipitation, desalted and applied to a MonoQ column. The column was washed with 5 column volumes of the equilibrating buffer. No detectable kinase activity was eluted in the flow-through and wash fractions. When the column was developed by using a linear salt gradient with NaCl over the range 0-0.5 M, three peaks of MBP-kinase activities eluted at approximately 0.2 M, 0.3 M and 0.42 M NaC1 as shown in Fig. 2. The comparable fractions from untreated tissue (no insulin) contained very little MBPkinase activity even though the amount of protein loaded onto the MonoQ column was very similar with the two different extracts. Thus, the stimulatory effect of insulin on the protein kinases was preserved during this chromatographic step. Determination of the protein concentration in the NaCl-eluted fractions indicated that the kinase activities eluted after the major protein peak.  54  FIGURE 2. MONOQ CHROMATOGRAPHY OF INSULIN-ACTIVATED SERINE/THREONINE KINASES FROM RAT ADIPOSE TISSUE.  High speed supernatant fractions were prepared from rat adipose tissue which had been treated with (10 mU/m1 for 10 min) or without insulin and were subjected to ammonium sulphate precipitation, desalted using a Bio-Rad Econo-Pac 10 DG desalting column and loaded onto a MonoQ column. The fraction applied (4 ml) contained 1 mg protein per ml. The column was developed with a 30-m1 linear 0-0.5 M NaC1 gradient. The column fractions were assayed for MBPphosphorylating activity.  ^ 0.5  100  75  • —• +insulin 0-0 -insulin  -0.4  •  0_ o^ _c^\ a^o  50  _c CL^0_^25 0_  0  -0.3  ** •  /  -0.2  ^••:\- - o •  • **  -••••••••••••••••!00?-2°°°°°°°°o°°°°" --------1-04t ^0.0 0^10^20^30^40 FRACTION #  O  55  3.1.2 DEAE-CELLULOSE ION EXCHANGE CHROMATOGRAPHY  High speed supernatants precipitated with 50% ammonium sulphate were desalted and applied to a DEAE column which was developed with a single step elution with buffer containing NaCl at 0.5 M. Protein kinase activity able to phosphorylate MBP eluted in a single sharp peak as shown in Figure 3. This column did not resolve the different MBP kinases present in the fat pad extracts but did achieve a rapid enrichment of kinases that was useful in some of the subsequent column procedures which were attempted - namely the use of phenyl-Sepharose, which requires the application of the protein fraction in the presence of high salt concentrations.  56  FIGURE 3. DEAE-CELLULOSE ION EXCHANGE CHROMATOGRAPHY OF INSULIN-ACTIVATED SERINE/THREONINE KINASES FROM RAT ADIPOSE TISSUE.  High speed supernatant fractions were prepared from rat adipose tissue which had been treated with (10 mU/m1 for 10 min) or without insulin and were subjected to ammonium sulphate precipitation (50% saturation). The pellets were re-dissolved, desalted using a Bio-Rad 10DG Econo Pac column and applied to a DEAE-cellulose column. The fraction applied (4 ml) contained 2 mg of protein per ml. After washing off unbound proteins, the column was eluted with 0.5 M NaC1 and 2-ml fractions collected. The figure below shows the protein kinase activity assessed by phosphorylation of MBP.  1000  800 0 2 w c  • —• +insulin 0-0 -insulin  a 600 c  O  E w 400 O _c a 0 m a o E 200 0  3^5  ^  FRACTION #  8^  9  57  3.1.3 PHENYL-SEPHAROSE CHROMATOGRAPHY.  In addition to ionic interactions, hydrophobicity was another physiochemical property of proteins which we considered might be exploited in the logical design of a purification scheme. The separation of proteins using hydrophobic interaction (HIC) is based on a different principle from most other separation techniques and can thus, in combination with these other methods, afford a high degree of purification. In addition HIC is generally a mild method due to the stabilizing influence of salts and recoveries are often high (Eriksson, 1989). It is a technique which has proved very valuable in other studies of mitogen-activated protein kinases. Rat adipose tissue MBP-kinase activity partially purified by DEAE-cellulose ion exchange chromatography was further fractionated using HIC using a phenyl-Sepharose column (2.5 x 6 cm). The sample was applied in a buffer containing 0.8 M ammonium sulphate to promote the hydrophobic nature of the proteins. The column was eluted stepwise using a sequence of buffers as follows: firstly, a buffer identical to the application buffer except for the omission of ammonium sulphate, secondly, the same buffer containing ethylene glycol (50%, w/v) and finally with buffer containing the detergent Nonidet P40 (NP40, 1.5%, w/v). Three peaks of kinase activities were eluted under these three different elution conditions as shown in Figure 4.  58  FIGURE 4. PHENYL-SEPHAROSE CHROMATOGRAPHY OF INSULINACTIVATED SERINE/THREONINE KINASES FROM RAT ADIPOSE TISSUE.  High speed supernatant fractions were prepared from adipose tissue which had been treated with or without insulin (10 mU/m1 for 10 min) and were then applied to a DEAE-cellulose column. Protein kinase activity was eluted with a singlestep buffer change (0.5 M NaC1). Ammonium sulphate (0.8M) was added to the pooled DEAE-cellulose fractions exhibiting MBP-phosphorylation activity. This pooled fraction 4 ml, containing 1.5 mg/ml protein was applied to a phenylSepharose column and eluted sequentially as follows: (a) buffer identical to the application buffer except for the omission of ammonium sulphate (fractions 1-10), (b) the same buffer containing 50% (v/v) ethylene glycol (fractions 1120) and finally (c) with buffer containing the detergent Nonidet P40 (1.5%, w/v; fractions 21-30).  40 >, ._ > .7 o ----. 30 w E o a La) 4-a)^cp_ -  c . c 20 E 2^ ,,  •  • —• +insulin 0-0 -insulin b  I  c  ,^ •^ o a.) Q_^ 17)  E 8 o.. 10 _c ,_, (  ci_ a_ co  • ^\• 0.  /  • / 0  ./\\^A^ / oor ,, 0^0  • ...,, 0,.. 0  •  I  0  0•• 00!(!lmf P••tp.,,,,000 ^ I 00 31 11^21 FRACTION #  59  3.2^PURIFICATION  ^  OF^INSULIN-ACTIVATED^PROTEIN  SERINEJTHREONINE KINASES USING SEQUENTIAL CHROMATOGRAPHY WITH POLYLYSINE-AGAROSE, MONO() AND HEPARIN-AGAROSE COLUMNS.  A high speed supernatant fraction was prepared from rat adipose tissue (obtained from 18 rats) which had been treated with insulin and this supernatant was precipitated with ammonium sulphate as described above. The protein pellet precipitated by ammonium sulfate (50% saturation) was resuspended, diluted to a volume of 200 ml and applied to a polylysine-agarose column (2.5 x 6 cm) which was developed with a linear salt gradient using NaCl over the range 0.20.6 M. Two broad peaks of MBP kinase activities eluted with peak maxima eluting at approximately 0.23 M and 0.43 M NaCl (Fig. 5). These peaks are now designated PLI and PLII respectively, (polylysine peaks I and II respectively). Pooled fractions from PLI and PLII were diluted and separately applied to a MonoQ column. The column was developed with a linear 0-0.8M NaCl gradient. In order not to exceed the capacity of the column, the pooled samples were applied in three separate runs and the column fractions collected in the same set of tubes three times. The elution profiles of the kinase activities are shown in Fig. 6. In both cases, sharp peaks designated MQI and MQII were obtained. MQI and MQII are the protein kinase activity peaks obtained by fractionating PLI and PLII, respectively, on the MonoQ column.  60  The kinase activity in MQII was more tightly bound to both the polylysine-agarose and the MonoQ columns. Based on western blotting analysis to be described in detail below, MQI was further purified using heparin-agarose and then a final MonoQ column fractionation (Fig. 7) and a silver stained gel of the final MonoQ preparation is illustrated in Fig. 8.  • 61 FIGURE 5. CHROMATOGRAPHY OF INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASES ON POLYLYSINE-AGAROSE.  High speed supernatant fractions from 25g insulin-treated tissue (10 mU/m1 for 10 min) were precipitated using ammonium sulphate (50% saturation). The pellet was resuspended, diluted to a volume of 200m1 and applied to a polylysine-agarose column which was developed with a linear salt gradient using NaCl over the range 0.2-0.6 M (dotted line). Four-milliliter fractions were collected and assayed for MBP-phosphorylating activity.  2000 N  O I  <  0.6 - 0.5  1500  0  }2  5 60' _5 = —Ec  1000  500 a_ •  •  •  /•  •  0  • fbio  • ^•^• • •• •  0  0  —0.4  • ^0  - 0.2 °  I^ 0.1 0^10^20^30^40^50^60^70^80  FRACTION #  0  62  FIGURE 6. CHROMATOGRAPHY OF INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASES ON MONO Q.  Active fractions containing MBP-kinase activity from the polylysine-agarose illustrated in Fig. 5 were pooled separately and applied to a MonoQ column. After washing off unbound protein, the column was eluted with a linear 0-0.8 M NaC1 gradient. Panel A, chromatography of pooled fractions (24-34) from polylysine peak 1; panel B, chromatography of pooled fractions (50-60) from polylysine peak 2. 2000 0  or° mI 2 107)^x  0.800  •  A  0.600  1500 -  c  2 E t -c^01" '  6  E  0 .c  m  a_  *  0.400 0  •1.  1000 —  z  • •• •  500 -  0.200  0 • ••••••••••••••••••• 0.000 0 ----.111=-11-1-1—!"• °^ 1 0 20 30^40^50^60^70^80 0  •  0 0  FRACTION #  ,  • 470  a) ca 2  q) •  0.800  2000  B - 0.600  ° 1500 x  c  2  o _c 0_ 0o.  m  • •  - 0.400  1000  E  a o  •  500  0  • 1 Pe ••  k••■••.•• ►^I .  0  - 0.200 0  •••••••♦••• • •••••• 0.000  10^20^30^40^50^60^70^80 FRACTION #  • 63  FIGURE 7. CHROMATOGRAPHY OF INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASES ON MONO Q.  Active fractions from sequential chromatography on polylysine-agarose (peak 1), MonoQ (Fig. 6, panel A), heparin-agarose (results not shown) were finally fractionated on MonoQ. The column was developed with a linear 0-0.8 M NaC1 gradient at a flow rate of 1 ml/min. Two hundred and fifty microliter fractions were collected and assayed for MBP-phosphorylating activity.  •  1500 0  0.800  w I O 0 w X 1000  0.600  C  2E O  0.400  S-  -C^(2) 0-  o_  o S = E 500 co 0_ o  •  •  8  (7) 0 0.200 00•• • 10^20  0 0  • 30^40^50 FRACTION #  60  70  80  0.000  64  FIGURE 8 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS OF PURIFIED INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASE.  One hundred and fifty microliters of each of the indicated fractions from the MonoQ column illustrated in Fig. 7 were precipitated with prechilled acetone (-20°C). The recovered protein pellets were dissolved in 200 pl SDS-digestion buffer and 40 pl of this sample electrophoresed on a gradient (7.5-15%) polyacrylamide gel. The gel was stained with silver nitrate. Lane 32 contained the most highly purified enzyme which corresponded with the peak of kinase activity. The arrow shows the position of the 44 kDa band. The position of the molecular weight markers (in kDa) is indicated.  30 200►  97►  46►  30■ 22 ►  ^ ^ 32 33^34 31  65  4.0 WESTERN BLOTTING ANALYSES.  Further characterization of the protein kinase activities separated on different columns was carried out using Western blotting analyses. Two of the antibodies used were raised against peptide antigens based on the primary sequences within different domains of the ERK1 kinase. Antiserum was raised against "R2" within the domain near the C-terminus of ERK1 while "R1" was directed towards a sequence surrounding kinase subdomain III of ERK1. The anti-p44 mP k antibody was raised against purified p44 mP k , a sea-star protein serine/threonine kinase which was shown to phosphorylate acetyl-CoA carboxylase apparently on the insulin directed site. (Pelech et al., 1991). This antiserum was subsequently purified by immunoaffinity chromatography. In addition, two other antibodies were used, one was a monoclonal antiphosphotyrosine antibody, the other was designated "GEGA" antibody. The GEGA antiserum is antiserum raised against a peptide in p44 mP k which corresponds to part of the ATP-binding region in conserved kinase subdomain I. The sequence is generally designated GXGXXG and forms part of the nucleotide-binding fold of all known protein kinases. It was anticipated that the use of these antibodies would ascertain whether homologs of ERKs and/or p44 mP k existed in extracts of insulin-stimulated rat adipose tissue extracts and the extent to which any immunoreactive bands copurified with insulin-stimulated protein kinase activity. In addition  66  the GEGA and phosphotyrosine antibodies would confirm whether the crossreacting proteins were kinases and/or tyrosine phosphorylated. Although fractions were analyzed for immunoreactive bands throughout all the gradients, many of the fractions which showed little or no reactivity are omitted for clarity.  4.1 IMMUNOBLOTTING WITH ANTIBODY RAISED AGAINST SEA-STAR D44 mP k .  The anti-p44 mP k antibody reacted with polypeptides of approximate molecular weights 40, 42, 44 and 70 kDa (Fig. 9). These peptides were well resolved during MonoQ anion exchange chromatography that was shown in Fig 2. The 44 kDa protein band in particular, copurified with the first peak of protein kinase activity from that column corresponding to fractions 22, 23 and 24. In contrast, the bands of 40 and 42 kDa emerged in fractions 25 26 and 27 with a peak around fraction 26 and the 70 kD band appeared last of all, in fractions 29,30 and 31. These observations suggest at least 3 protein kinase activities which are separable by Mono Q, which are all related in some way (immunologically) to p44 mP k and which display distinct subunit Mr values.  67  FIGURE 9. CHROMATOGRAPHY OF INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASES ON MONO Q AND IMMUNOBLOTTING WITH VARIOUS ANTIBODIES.  Chromatography was performed as described in Fig. 2. One hundred microliter samples from each fraction were precipitated with prechilled acetone (-20°C) and the recovered protein pellets dissolved in 40 Al SDS-digestion buffer. These were then electrophoresed on gradient (7.515%) polyacrylamide gels. The separated proteins were then transferred to immobilon membrane. Finally, the blots were probed with the anti-p44 mP k (p44), antiphosphotyrosine (PY), R2, R1 and GEGA (GE) antibodies as shown below. Arrows 1 and 2 indicate the positions of the 44 and 42 kDa bands, respectively. The bars show fractions within the peaks of MBP-kinase activity. The position of the molecular weight markers (in kDa) is indicated. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 n 39 40 41  68  When fractions from phenyl-Sepharose column were probed with anti-p44 m1* antibody, all the above mentioned polypeptides were detected. Most, if not all immunoreactive protein bound to the phenyl-Sepharose and very little emerged in the void fraction. Indeed, very little immunoreactive protein emerged in the wash treatment using low salt buffer. When the column was eluted with 1.5% Brij 35, a single peak of protein kinase activity was recovered. All the immunoreactive polypeptides copurified within this peak of kinase activity. In contrast when the column was eluted sequentially with 50% ethylene glycol and subsequently with 1% NP40, separation of these polypeptides was achieved. The 44 kDa as well as a faint 42 kDa band were detected within the ethylene glycol eluant. The 70 kD polypeptide was identified within the fractions eluted with the detergent. It is therefore clear that the immunoreactive proteins and insulin-stimulated protein kinases do indeed bind very tightly to phenyl-Sepharose. Specific elution conditions can be developed to achieve some separation of the different immunoreactive polypeptides recognized by anti-p44 mPk antiserum. It is worth pointing out that even if this latter resolution is not achieved, the phenyl-Sepharose column nevertheless, provides considerable purification due to the removal of a large amount of unbound protein in the void fraction combined with the low-salt wash.  69  Polylysine-agarose chromatography resolved two peaks of kinase activities as already described. The first peak was shown to contain a 44 kDa polypeptide which crossreacted with p44 mP k (Fig. 10). Consequently, the polylysine-agarose fractions containing the kinase activity within the first peak were subjected to further fractionation employing three consecutive column steps - MonoQ, heparin-agarose (results not shown) and finally a second MonoQ column. The antip44 mP k antibody recognized a 44 kDa protein band which copurified with protein kinase activity (Fig. 11) throughout these three further purification steps. The second polylysine peak contained immunoreactive polypeptides of approximate molecular weight 65 and 75 kD which reacted with the anti-p44 mP k antibody (Fig. 12). Based on these results, polylysine-agarose fractions from the second peak containing protein kinase activity were not further purified, at the present time although this will be of interest in future studies.  70  FIGURE 10. IMMUNOBLOTTING OF INSULIN-ACTIVATED RINASES PARTIALLY PURIFIED ON POLYLYSINE-AGAROSE.  Pooled fractions from the first peak of polylysine chromatography were further fractionated on MonoQ as shown in Fig. 6A. Two hundred microliter samples from each of the indicated fractions were precipitated with 1 ml of prechilled (-20°C) acetone. The recovered pellets were dissolved in SDS-digestion buffer and electrophoresed on gradient (7.5-15%) polyacrylamide gels. The separated proteins were transferred to immobilon membrane. The blots were probed with the anti-p44n k (p44) and R2 antibodies as shown below. The peak of MBP-kinase activity was in fraction 32 (Fig. 6A). The position of the molecular weight standards (s) in kDa is indicated.  24 25 2627 28 29 30 31 32 33 34 35 4 97  p44  446 430  30 31 32 33 S  R2  197 146 130  71  FIGURE 11. IMMUNOBLOTTING OF PURIFIED INSULIN-ACTIVATED PROTEIN SERINE/THREONINE KINASE.  High speed supernatant fractions from insulin-treated adipose tissue (10 mU/ml for 10 min) were subjected to sequential purification steps to enrich for insulinstimulated MBP-kinase. The steps involved were: polylysineagarose (Fig. 5), MonoQ (Fig. 6A), heparin-agarose (results not shown) and MonoQ (Fig. 7) and the results of the final MonoQ step are illustrated here. One hundred and fifty microliter volumes from each of the indicated fractions from the final MonoQ column were precipitated with 1 ml of prechilled (-20°C) acetone. The precipitated proteins were dissolved in 200 Al of SDS-digestion buffer and 40A1/lane electrophoresed on gradient gels (7.5-15%). The separated proteins were then transferred to immobilon and probed with the anti-p44 mP k (p44), R1, R2, antiphosphotyrosine (PY) and GEGA (GE) antibodies as shown below. The position of the molecular weight standards (S, in kDa) is also indicated.  S 12 13 14 15 16 S 30 31 32 33 34 200 97 46  p44  ■ 30 ■ 14  -4 200  ■ 97 ■ 46 -4  30  -4114  R1 R2 PY GE S  72  FIGURE 12. IMMUNOBLOTTING OF FRACTIONS FROM POLYLYSINE PEAK 2.  High speed supernatant fractions from insulin-treated adipose tissue (10 mU/ml) were applied to polylysine-agarose and fractions from peak 2 were pooled and further fractionated on the MonoQ as shown in Fig. 6B. Two hundred microliter samples from the latter purification step were precipitated with 1 ml of prechilled acetone (-20°C). The recovered proteins were dissolved in 200 gl of SDS-digestion buffer. Forty microliter samples from each of the indicated fractions were electrophoresed on polyacrylamide gels and subsequently transferred to immobilon membranes. The blots were probed with the anti-p44 mP k (p44) and R2 antibodies as shown below. The position of the molecular weight markers (in kDa) is indicated.  28 29 30 3132 33 34 35 36 3738 39 40  ANTI-p44  35 36 37 38 39 40  R2^  1 97 has 130  73  4.2 IMMUNOBLOTTING WITH R2 ANTIBODY.  R2 antibody reacted very strongly with a 40/42 kDa doublet in protein fractions separated on the MonoQ column (Fig 9). It appeared not to crossreact with the 44 kDa band which was detected using anti-p44 mP k antibody. Furthermore, this doublet was well resolved during the denaturing SDSPAGE analysis from the 44 kDa band. Perhaps paradoxically, the R2 antibody did appear to react with the 44 polypeptide which copurified with protein kinase activity which emerged in the earlier of the two peaks observed during polylysineagarose chromatography. Therefore, I obtained apparently different reactivity between the fat pad 44 kDa band and the anti-p44 mP k antibody depending upon the purification step used.  4.3 IMMUNOBLOTTING WITH Rl ANTIBODY.  The Ri antibody also reacted with the 42 and 40 kDa polypeptides in protein fractions separated by Mono Q ion exchange chromatography (Fig. 9). This doublet was especially prominent in fractions 25 and 26. Incidentally, these are the same fractions where the 40/42 kD doublet reacted very strongly with R2 antibody. In contrast to R2, R1 reacted with a 70 kD band which was also detected by anti-p44 mP k antibody. As with the R2 antibody, Ri reacted  74  with the 44 kDa band in the most highly purified fraction containing protein kinase activity (Fig. 11).  4.4 IMMUNOBLOTTING WITH ANTIPHOSPHOTYROSINE ANTIBODY.  Since other MAP kinases have been shown to be regulated by tyrosine phosphorylation, probing with anti-phosphotyrosine antibodies was an important avenue to persue. When protein fractions separated by MonoQ anion exchange chromatography were probed with the antiphosphotyrosine antibody, only the 44 kDa and the upper band of the 42 kDa doublet were detected (Fig. 9). In contrast, neither the 40 kDa nor the 70 kDa bands showed any appreciable labelling in response to anti-phosphotyrosine antibodies. The antiphosphotyrosine antibody also reacted with the 44 kD band in the most highly purified fraction containing protein kinase activity (Fig. 11) showing that the tyrosine phosphorylation had been preserved at least partially throughout the extensive purification.  4.5 IMMUNOBLOTTING WITH GEGA ANTIBODY.  The GEGA antibody crossreacted with the 42 kDa doublet and to a lesser extent with the 44 kDa band in MonoQ fractions (Fig. 9). It also reacted with a 44 kDa band in the purified fraction containing protein kinase activity (Fig. 11). It is  75  surprising that the 70 kDa band apparently reacted only weakly, if at all, with the GEGA antibody. Nevertheless, the cross reactivity of the 44 kDa band with anti-p44 mP k , antiphosphotyrosine and GEGA argue strongly that the most highly purified preparation does indeed contain an insulinstimulated protein ser/thr kinase highly-related to other MAP kinases.  76  5.0 ADDITIONAL CHARACTERIZATION OF RAT ADIPOSE TISSUE SER/THR KINASES.  5.1 INHIBITION OF ENDOGENOUS PROTEIN PHOSPHORYLATION BY ADDED MYELIN BASIC PROTEIN.  High speed supernatant fractions prepared from fat pads treated with insulin, were incubated with [gamma- 32 P]ATP in the presence or absence of purified myelin basic protein. The phosphoproteins were separated by SDS-PAGE which was followed by autoradiography. As shown in Figure 13, addition of MBP specifically decreased  32 P-incorporation  into ATP-  citrate lyase (ATP), another insulin-directed protein, suggesting a possible interaction with the ACL-kinase. In these experiments, very little competition with acetyl-CoA carboxylase was observed. In a number of previous experiments, employing other protein kinase substrates (including histones and casein) no such interactions were observed. Furthermore, when bradykinin (a model substrate for proline-directed protein kinases) was used instead of 32 MBP P-incorporation into ACL was not inhibited.  77  FIGURE 13. INHIBITION OF PROTEIN PHOSPHORYLATION IN HIGH SPEED SUPERNATANT FRACTIONS FROM RAT ADIPOSE TISSUE BY ADDED MYELIN BASIC PROTEIN  High speed supernatant fractions from insulin treated fat pads (10 mU/m1 10 miA) were i ncubated (10 min., 30 C) in the presence of [gamma- J4 P]ATP-Mg with (lanes 1,3) or without (lanes 2,4) added myelin basic protein (5mg/m1). The proteins were precipitated with acetone and separated by SDS-PAGE, followed by autoradiography. The figure shows the coomassie blue stained gel (A) and the autoradiogram (B). The position of the molecular weight standards as well as that of acetyl-CoA carboxylase (ACC) and ATP-citrate lyase (ACL) are indicated.  A  78  5.2 IMMUNOPRECIPITATION OF MBP-KINASE ACTIVITY.  Attempts were made to immunoprecipitate MBP-kinase using R2 and anti-p44 mP k antibodies. Very little apparent decrease in supernant MBP-kinase activity was observed. The immunoprecipitates were analyzed by SDS-PAGE and showed a multitude of bands. It was therefore, difficult to draw any definitive conclusions. Another attempt at immunoprecipitating MBP-kinase was made using anti-phosphotyrosine antibodies. Only partial precipitation of polypeptides of subunit molecular mass 45, 42 and 30 kDa was achieved. When the immunoprecipitates were blotted with anti-p44 mP k polypeptide bands of subunit molecular weight of 45, 42 and 30 kDa were recognized. The R2 antibody reacted with the 42, 40 and 30 kDa bands. This observation lends further support to the idea that the 44 kDa band reacts weakly with R2 antibody compared to 42 and 40 kDa bands.  79  DISCUSSION.  Within seconds after binding of insulin at the cell surface, the B-subunit of the insulin receptor displays increased auto-phosphorylation on tyrosine residues. Autophosphorylation leads to enhanced protein-tyrosine kinase activity towards other cellular proteins - the functions of which are still being actively persued. Following these initial events, a wide range of subsequent responses occur within target cells. Among these subsequent effects of insulin action are rapid increases in phosphorylation of serine and/or threonine (ser/thr) residues on target proteins like ACC, ACL and the S6 ribosomal protein. These changes in protein phosphorylation occur as a result of the activation of proteinserine/threonine kinases which appear to serve as critical signaling intermediates, and as such their identification and characterization is important in the elucidation of the multiple steps of the pathway, initiated by receptor activation. Studies of insulin-activated ser/thr kinases has been technically simplified by using substrates which are more convenient than the anticipated more physiological substrate enzymes themselves, which must be isolated usually by laborious, lengthy procedures. In addition, these cellular substrate enzymes often (like acetyl-CoA carboxylase, of interest in these studies) contain multiple phosphorylation  80  sites which mean that the determination of sites phosphorylated would require additional 2-dimensional phosphopeptide analysis. This would be extremely timeconsuming and really not feasible as a routine assay to be used for following kinase activity during purification procedures. In these studies, myelin basic protein (MBP) was used as a model substrate for the insulin-activated kinases. After initial studies with MBP purified by another research group at UBC (Dr. Myer Bloom, Physics), MBP has become commercially available (Sigma). MBP binds strongly to P-81 phosphocellulose paper and therefore is ideally suited for filter paper assays. This choice was justified because MBP appears to be an excellent substrate for mitogen-stimulated kinases in a number of studies and indeed was found to successfully detect insulin-activated protein kinase in extracts of rat adipose tissue. Furthermore, a MBP-kinase from sea-star was shown to phosphorylate acetyl-CoA carboxylase on the insulin-directed site. The tissues most commonly used to study insulin effects are muscle, liver, adipose and lactating mammary glands which are all sensitive to the hormone. Of the four tissues, adipose tissue can be most easily manipulated, in vitro and shows the most striking changes in metabolism following in  vitro incubations with insulin. The major points addressed in these studies were preserving the insulin-activated  81  Ser/Thr kinases from adipose tissue, and then subsequent purification and characterization using various antibodies.  STABILIZATION OF INSULIN-ACTIVATED MBP-KINASES.  Initial experiments to study insulin-stimulated Ser/Thr kinases were carried out using freshly-prepared high speed supernatant fractions. Further progress in the purification and characterization of these kinases of interest was hampered by the rapid loss in enzyme activity upon storage. The insulin-stimulated Ser/Thr kinases proved to be labile under many commonly used storage conditions such as at 0°C, 4°C, -20°C or -70°C and despite the use of stabilizing agents such as glycerol, proteinase inhibitors and certain phosphatase inhibitors. The immediate concern was therefore, to try to stabilize these insulin-activated protein Ser/Thr kinases. This was ultimately achieved by employing a rapid procedure which included ammonium sulphate precipitation and which stressed the use of a cocktail of phosphatase inhibitors in the extraction buffer. Several advantages are achieved by using ammonium sulphate precipitation. Firstly large sample volumes could be employed and dual control and insulin-treated samples easily treated in parallel. Secondly, the resulting pellets could be dissolved in a small amount of buffer, thus increasing the protein concentration of the sample and probably aiding in stabilization. Finally, the high salt  82  concentration employed is often inhibitory to enzyme action, therefore, the precipitated proteins could be stored in a stable form with likely minimal proteolytic action. In addition, to these benefits of rapid precipitation with ammonium sulfate, the stability of the insulin-activated protein kinases was aided by the presence of phosphatase inhibitors in the extraction buffer. In particular, a turning point seemed to be the adoption of a particularly potent inhibitor of protein phosphatases I and 2A, namely microcystin. This observation reinforces the possibility that the insulin-activated kinases may in turn be regulated by phosphorylation, a feature which has certainly emerged as being highly significant in the studies of other so-called "mitogen-activated kinases".  PURIFICATION OF INSULIN-ACTIVATED MBP-KINASES.  Having now overcome the problem of instability of the kinases the next priority was purification and characterization of the insulin-stimulated Ser/Thr kinases. Purification of the polylysine MBP-kinase was achieved using ammonium sulfate precipitation followed by sequential chromatography on polylysine-agarose, MonoQ, heparin-agarose and finally Mono Q for a second time. The silver-stained gel of the final purified fraction contained a major 44 kDa band which copurified with the peak of MBP-kinase activity.  83  Assignment of this protein band as a kinase was supported by its comigration with Ser/Thr kinase activity, immunoreactivity with antibodies which recognize ERK1 and p44 mP k kinases. In addition the immunoreactivity of the 44 kDa band with the GEGA antibody in particular, lends further support to this conclusion, since it recognizes one of the highly conserved regions involved in ATP-binding found in all known kinases and which includes the concesus GXGXXG motif. The preparation of the insulin-activated protein serine/threonine kinase so far achieved is not homogenous. One major (high molecular weight) band and several minor bands are still present and may need to be removed. However, based on immunoreactivity with the various antibodies as well as the homogeneity of chromatographic peaks of kinase activity, it seems unlikely that any other protein kinases remain in the preparation. Significantly, the gel is remarkably "clean" below 44 kDa, suggesting the complete removal of p42 and other lower molecular kinases. Though not absolutely homogenous, this preparation may yet be sufficient for detailed analysis. It will be necessary to confirm that the 44 kDa band is itself a single protein by running 2-dimensional gel analysis. If indeed this is the case, then the preparation can immediately be scaled up to attempt protein sequencing directly from the gel. Another graduate student in our lab (Bob Winz) has already achieved this for another protein while working under the guidance of Dr. Aebersold (BRC) and  84  so we are fortunate that these techniques are available. This is perhaps the single most critical objective. Following either N-terminal or partial internal sequencing, all the necessary information would be available to initiate molecular cloning. This would therefore, allow us to unambigously establish the relationships of this important kinase to other members of the family.  RELATIONSHIP OF ADIPOSE TISSUE P44 MBP-KINASE TO OTHER INSULIN-STIMULATED PROTEIN SERINE/THREONINE KINASES.  The p44 kDa MBP-kinase is clearly distinct from the S6 kinases by virtue of its molecular weight and substrate preference. The S6 kinases are larger enzymes, exhibiting subunit molecular weights of around 67-70 and 90 kDa under denaturing conditions employed for SDS-PAGE, contrasting with 44 kDa for MBP-kinase. In addition, chromatographic properties of these enzymes are different. S6 kinases bind avidly to anion exchangers and therefore require higher salt concentrations for elution. Furthermore, S6 kinases bind to heparin columns whereas, the p44 MBP-kinase described here does not bind at all to such a column. An insulin-stimulated protein-serine kinase (Kemptide kinase) from the cytosol of rat adipocytes has been reported by Czech and coworkers (Klarlund et al., 1990). However, the insulin-stimulated ser/thr kinase reported in this study appears to be unrelated to this cytosolic kinase. This is  85  because of the difference in molecular weights. The MBPkinase has a subunit molecular size of 44 kDa whereas, that of the Kemptide kinase (KIK) is 52 kDa. With respect to substrate specificity, KIK exhibits preference to histone, kemptide and ACL. The site phosphorylated in kemptide has the sequence LRRASLG which is different from the substrate concensus sequence of MAP kinases. The latter phosphorylate Ser/Thr residues in close proximity to proline residues such as Thr 97 in bovine MBP (PRTPPP, Erickson et al., 1990; Sanghera et al., 1990a; Clark-Lewis et al., 1991). In light of the close resemblance of p44 MBP-kinase to other MAPkinases, it is more likely that p44 MBP-kinase preferentially recognizes sequence motifs with proline determinants. Another striking difference between KIK and MBP-kinase is that the latter reacts with antiphosphotyrosine antibodies whereas, KIK lacks this immunoreactivity. A further distinguishing feature between p44 MBP-kinase and KIK is the ability of KIK to bind to heparin columns. Casein kinase II (CKII) is another Ser/Thr kinase reported to be stimulated by insulin. However, p44 MBPkinase differs markedly in both substrate specificity and structural properties from CKII which exists as a heterotetramer of a and B subunits. CKII is "acidotropic", in that it phosphorylates sites in motifs containing either Glu, Asp or Ser(P) such as in the CKII peptide (-EEETEEE; Roach, 1991).  86  From the evidence we so far have available, it seems likely that p44-MBP-kinase is related to a family of protein Ser/Thr kinases known as ERKs or MAP kinases. This resemblance is based on a range of properties including immunological, chromatographic, subunit molecular weight and phosphorylation on tyrosine residues. Similarities between p44 MBP-kinase and the recently purified ERK1 kinase include substrate specifity, molecular weight and crossreactivity with R1, R2, anti-p44mPk and anti-phosphotyrosine antibodies. However, a subtle difference existed when R2 and R1 antibodies (raised against peptides patterned against the C-terminal and subdomain III regions in ERK1) were used to probe blots which contained the 44, 42 and 40 kDa polypeptide bands. Only the 42 and 40 kDa bands were recognized. Surprisingly, these antibodies appeared not to react with the 44 kDa band corresponding to MBP-kinase. It is possible that p44 MBP-kinase contains sequence differences that reduce its recognition by R2 antibody. In light of this, it would appear that ERK1 is more closely related to the 42 and 40 kDA polypeptides than it is to p44 MBP-kinase. A definitive assignment of how much ERK1 and p44 MBP-kinase are related  however,^awaits^further  characterization of MBP-kinase particularly determination of the primary sequence. Other members of the ERK family include ERK2 and ERK3. The predicted molecular weights of these kinases are 41.2 for ERK2 and 62.6 kDA for ERK3, contrasting with 44 kDa for  87  MBP-kinase. It is therefore unlikely that the MBP-kinase corresponds to either ERK3 or ERK2. In addition to the ERKs which are derived from molecular cloning using a rat brain cDNA library, other related kinases have been described, mostly using murine cells. The now-classical studies of Ray and Sturgill reported an insulin-activated ser/thr kinase from 3T3-L1 cells (p42 ma P k ), recognized by the ability to phosphorylate MAP-2. This kinase has a subunit molecular weight of 42 kDa and is highly related to the ERKs, most likely the murine equivalent of ERK2. The published amino acid sequence of the murine p42 ma P k (Her et al., 1991) is identical to that of ERK2 (Boulton et al., 1991) which supports the idea that p42 ma P k is the murine equivalent of ERK2. There also appears to be a murine kinase with subunit molecular weight of 44 kDa which is highly related to the sea-star p44 mP k and presumably very closely related to the rat enzyme studied here. Similar "pairs" of kinases of subunit molecular weight 44 and 42 kDa have been recognized in murine 3T3 cells in response to EGF (Ahn and Krebs, 1990) and in growth-factor stimulated KB cells. In the latter case, the stimulated kinase activity was shown to account for most of the phosphorylation of the EGF receptor (Northwood et al., Alvarez et al., 1991). Studies with growth-factor stimulated Swiss 3T3 cells have also demonstrated the existence of two RSK protein kinases. Both RSK kinase I (44 kDa) and II (42  88  kDa) are related to sea-star p44 mP k and can partially activate p9O rsk in vitro. Together, these findings are consistent with the existence of a family of mitogen activated protein-serine/threonine kinases. Further progress toward the identification of the relationships between these enzymes will require the molecular cloning of all of these family members. Overall, one may deduce that the family of ERK/MAP kinases is complex. At least there appear to be distinct 44 and 42 kDa proteins (and these differ somewhat between species as well as within species). In addition, we also observe a 40 kDa band, so that we cannot tell yet the relationship of the ERKs with any of the kinases in rat adipose tissue.  INHIBITION OF ENDOGENOUS PROTEIN PHOSPHORYLATION BY ADDED MYELIN BASIC PROTEIN.  Initial "competition" studies using purified MBP as a competitive substrate in high speed supernatant fraction incubations indeed suggested a decline of phosphorylation of ACL and to a lesser extent ACC. Overall, however, after several more experiments it seems this effect is not striking. We have no clear understanding why some results were more positive than others. The only real difference was in the species of origin of MBP, being human (supplied by Frank Nezil, Physics Dept., UBC) versus commercial bovine  89  MBP. It is possible that some inter-species differences in primary sequence might explain the different results.  IMMUNOPRECIPITATION.  Attempts at immunoprecipitation of MBP-kinase from highspeed supernatant fractions with R2 and anti-p44 mP k was not successful. It is possible that these antibodies have a low affinity for the MBP-kinase and therefore could not successfully precipitate the kinase. Certainly, it is not unusual for antibodies to successfully recognize antigens on Western blotting while producing little or no immunoprecipitation of native antigen from solution. Presumably, critical epitopes are exposed following denaturation. In addition, if the antibody/antigen complexes were formed as a result of single point attachments rather than multimeric, then immunocomplexes would have easily remained in solution. This also suggests that for immunoaffinity purification of active kinase, the antiphosphotyrosine antibodies would be the best of the presently available choices. Unlike R2 and anti-p44 mP k antibodies, the antiphosphotyrosine was able to precipitate polypeptides of subunit molecular weight 44, 42, and 40 kDa. However, there was only partial recovery of these bands in the immunoprecipitates in comparison to the residual supernatants. This would be consistent with either partial  90  tyrosine phosphorylation, weak immunoreactivity or both of these phenomena. Since several different antiphosphotyrosine antibodies are commercially available, it would be worth checking the merits of each of them.  TIME-COURSE/DOSE RESPONSE FOR MBP-KINASE ACTIVATION.  Trials to determine the kinetics of activation of the MBP-kinase in response to insulin have been carried out but further work is required. High speed supernatant fractions of rat adipose tissue contain several kinases able to phosphorylate MBP. It is therefore difficult to determine the contribution made by the specific insulin-activated kinases of interest with a simple assay procedure. It is important to stress that a rapid and simple procedure is necessary to facilitate determination of activation of MBPkinase in the multiple fractions generated during a timecourse or dose-response study. In order to circumvent this problem Western blotting analysis using anti-phosphotyrosine antibodies was undertaken. This was based on the assumption that activation of the MBP-kinases is accompanied by tyrosine phosphorylation. The results obtained following gel analysis of proteins in high speed supernatant fractions were still complex and it was difficult to draw any firm conclusions. These experiments however, helped us to confirm that the 44 kDa protein band is indeed a significant tyrosine-phosphorylated protein in insulin-treated fat  91.  tissue. When both^high speed supernatant and membrane fractions were immunoblotted with the anti-phosphotyrosine antibodies, a "rich" assortment of bands was highlighted, suggesting miltiple proteins became tyrosine-phosphorylated in response to insulin. A particularly, prominent 30 kD protein band was labelled with anti-phosphotyrosine antibody. This intriguing, major band remained in the ammonium sulphate supernatant when the high speed supernatant extract was subjected to ammonium sulphate precipitation (50% w/v). Since this band was also immunoreactive when tested with R2 and anti-p44 mP k antibodies, it would appear to be itself a protein kinase perhaps, related to p34 cdc2 , a protein-serine/threonine kinase that functions in the M-phase of the cell cycle. It is clear, therefore, that to accurately follow the dose- and time-dependency of p44 tyrosine phosphorylation, purification beyond the high speed supernatant or even ammonium sulphate precipitation will be required. The patterns are still too complex at this stage for easy interpretation. It seems that rapid phenyl-Sepharose purification will be one important possibility to be tested, as used by Anderson and coworkers (1991). Further, these studies have opened up an important avenue for further investigation namely the apparent complexity of the phosphotyrosine profile, the preponderance of membrane proteins affected and the striking effect on a 30 kDa protein.  92 CONCLUSION.  The present study has demonstrated the presence of an insulin-stimulated protein-Ser/Thr kinase in rat adipose tissue. The MBP-kinase is very similar to other mammalian mitogen-activated kinases in terms of molecular weight, immunological properties and substrate preference. This suggests that the MBP-kinase studied here in response to insulin belongs to the same family of Ser/Thr kinases described as MAP kinases. 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