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Structural and functional characterization of the Fujinami sarcoma virus transforming protein Weinmaster, Geraldine Ann 1985

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STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE FUJINAMI SARCOMA VIRUS TRANSFORMING PROTEIN by GERALDINE ANN WEINMASTER B.Sc., University of Saskatchewan, 1979 M.Sc, University of Saskatchewan, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standards University of British Columbia June, 1985 ®Geraldine Ann Weinmaster, 1985 In presenting t h i s thes is i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ib ra ry s h a l l make i t f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of t h i s thes is for scho lar l y purposes may be granted by the head of my department or by h i s or her representat ives . I t i s understood that copying or pub l i ca t ion of t h i s thes is for f i n a n c i a l gain s h a l l not be allowed without my wr i t ten permission. The Univers i ty of B r i t i s h Col 1956 Main Mall Vancouver, Canada V6T 1Y3 Department of D a t e ^)lJLfoJL. U s l<jf& ABSTRACT The phosphorylation of the Fujinami sarcoma virus transform-ing protein (FSV P 1 4 0 g a g - f p s ) is complex, reversible and affects i ts tyrosine specific protein kinase activity and transforming function. The sites of phosphorylation within FSV pi40 g a S~ f P s have been localized to various regions of the protein using partial proteolysis. The two major phosphotyrosine residues and a major phos-phoserine residue are located in the C-terminal portion of the fps region, which contains the kinase active domain. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three FSV variants shows that the phosphotyrosine containing peptides have similar mobilities. To determine whether tyrosine phosphorylation affects protein function and to evaluate the substrate specificity of the protein kinase intrinsic to FSV P130gag-fps oligonucleotide-directed mutagenesis was used to change tyrosine-1073, the major site of P 1 3 0 g a g - f p s phosphorylation. Tyrosine-1073 was mutated to a phenylalanine and a glycine, amino acids that cannot be phosphorylated, and to the other commonly phosphorylated hydroxyamino acids, serine and threonine. Neither serine nor threonine were phosphorylated when substituted for tyrosine-1073 indicating a str ict specificity for tyrosine. A l l of the FSV tyrosine-1073 mutants had depressed enzymatic ( i i ) and oncogenic capacities. These data indicate that tyrosine phosphory-lation stimulates the biochemical and biological activities of FSV PISO^8^ -^^8 and suggest that tyrosine phosphorylation modulates protein function. Mutations within the putative ATP-binding site of PISO^ 8^ -^ 8 at lysine-950 destroy both i ts kinase and transforming activit ies, supporting the idea that the tyrosine kinase activity intrinsic to P130^a^~^Ps is essential for i ts transforming function. The mutant protein was also shown to be phosphorylated at a second tyrosine site, which has been previously identified in wild-type PISO^3^ -^^ as a site exclusively phosphorylated in vivo. Phosphorylation of secondary tyrosine residues within a mutant protein devoid of intrinsic tyrosine protein kinase activity suggests that the FSV P 1 3 0 g a g - ^ p s may be a target for phosphorylation by1 cellular tyrosine specific protein kinases. ( i i i ) TABLE OF CONTENTS Page Abstract . . . . . . . i i List of Tables ix List of Figures . . . . . x List of Abbreviations x i i i Acknowledgments xvi Dedication xvi i CHAPTER 1 1.0 Introduction 1 1.1 Classification of Retroviruses 1 1.2 Structure of Retroviruses 1 1.3 Replication of Retroviruses 2 1.4 Oncogenic Retroviruses 3 1.4.1 Onc+ Oncogenic Retroviruses 3 1.4.1.1 Transduction of Cellular Oncogenes . . 4 1.4.2 One- Oncogenic Retroviruses 5 1.5 One and Proto-onc genes 6 1.6 Retroviral Oncogene Products 7 1.6.1 Tyrosine Kinase-Negative Class of Oncogene Proteins 8 1.6.2 Tyrosine Protein Kinase-Positive and Kinase-Related Class of Oncogene Proteins 10 1.7 Cellular Tyrosine Protein Kinases 16 1.8 Cellular Substrates of Tyrosine Kinases 18 1.8.1 Indirect Cellular Substrates of Tyrosine Kinases 25 1.9 Characteristics of Tyrosine Protein Kinases 26 1.10 Phosphorylation of Tyrosine Kinases - A Possible Role in Regulating Activity 29 1.10.1 Tyrosine Phosphorylation and the Regulation of Kinase Activity 29 1.10.2 Serine and Threonine Phosphorylation and the Regulation of Kinase Activity 31 (iv) Page 1.11 Fujinami Avian Sarcoma Virus . . . . 33 1.11.1 The FSV Genome 34 1.11.2 Variant Strains of FSV 35 1.11.3 The Relationship of FSV to Other Oncogenic Viruses 35 1.11.4 The FSV Encoded Transforming Protein . . . . 37 1.11.5 Cellular Location of the FSV Transforming Protein 39 1.11.6 The Normal Cellular fps Homologue 40 1.11.7 Structure of the FSV Gene Product 41 1.12 Purpose and Experimental Approach 44 CHAPTER 2 2.0 Materials and Methods 47 2.1 Cells and Viruses 47 2.2 Radiolabelling of Cells 48 2.3 Immunoprecipitation 49 2.4 Immune Complex Kinase Reaction 49 2.5 SDS-Polyacrylamide Gel Electrophoresis 50 2.6 Partial Proteolytic Cleavage with pl5 and V8 Protease 52 2.7 Analysis of Tryptic Peptides 53 2.8 Analysis of Phosphoamino Acids 56 2.9 Transfection of DNA into Rat-2 Cells 58 2.10 Oligonucleotide-directed Mutagenesis 60 2.11 Synthesis of p-Fluorosulfonylbenzoyl-51-Adenosine . . 62 2.12 Reaction of FSBA with P140gafi~fPs 62 (v) Page CHAPTER 3 3.0 Mapping of Multiple Phosphorylation Sites Within the Structural and Catalytic Domains of the Fujinami Avian Sarcoma Virus Transforming Protein 63 3.1 Introduction 63 3.2 Results 65 3.2.1 Tryptic Phosphopeptides of Pl40gag-fps 6 5 3.2.2 Localization of Phosphorylation Sites on P140gag-fps 70 3.2.3 Phosphorylation of the Transforming Proteins of Different fps Viruses 86 3.3 Discussion 92 CHAPTER 4 4.0 Oligonucleotide-direct Mutagenesis of Fujinami Sarcoma Virus: Evidence the Tyrosine Phosphorylation of P130Sag-fps Modulates i ts Enzymatic and Biological Activities 96 4.1 Introduction 96 4.2 Results 98 4.2.1 Oligonucleotide-directed Mutagenesis of FSV . . 98 4.2.2 Synthesis of a Revertant FSV 104 4.2.3 Transforming Activity of Wild-type, Mutant and Revertant FSV DNAs 107 4.2.4 Expression, Structure and In Vitro Kinase Activities of Wild-type and Mutant P130gag-fps Proteins I l l 4.2.5 Tyrosine Phosphorylation in wt FSV and FSV-F(1073) Transformed Cells 121 4.3 Discussion 128 (vi) Page CHAPTER 5 5.0 The Protein Kinase Activity of FSV P 1 3 0 g a g - f p s Shows a Strict Specificity for Tyrosine Residues 133 5.1 Introduction 133 5.2 Results 135 5.2.1 Oligonucleotide-directed Mutagenesis of FSV. . . 135 5.2.2 Transforming Activity of Wild-type, Mutants and Their Revertant FSV DNAs 140 5.2.3 Expression, Structure and In Vitro Kinase Activities of wt and Mutant Proteins 143 5.2.4 Tyrosine Phosphorylation of wtFSV and Mutant FSV PlSOg^-fps from Transformed Cells 150 5.3 Discussion 155 CHAPTER 6 6.0 Site-directed Mutagenesis of Lysine-950 Within P 1 3 0 g a g _ f p s Eliminates Both Its Kinase Activity and Transforming Ability 159 6.1 Introduction 159 6.2 Results 164 6.2.1 Inactivation of P140SaS- fP s Kinase Activity by Treatment with FSBA 164 6.2.2 Site-directed Mutagenesis of Lysine-950 Within the Putative ATP-binding Site of PlSOg^-fP 8 . . 169 6.2.3 Transfection of Rat-2 Cells with Wild-type, Mutant and Revertant FSV DNAs 172 6.2.4 Expression and Characterization of Mutant FSV-R(950) and FSV-G(950) P130e a&- fP s Proteins. . 176 (vii) Page 6.2.5 Trans-phosphorylation of FSV-R(950) Pl30gag~fps by FSV P140Sag~fPs 184 6.3 Discussion 190 CHAPTER 7 7.0 Summary 1 9 6 References 2°0 ( v i i i ) LIST OF TABLES Page Table 1.1 Retroviral Oncogenes 9 Table 1.2 Avian Sarcoma Viruses, Their Cell-derived Sequence Inserts and Gene Products 36 Table 4.1 Quantitation of the Kinase Activities of P 1 3 0 gag-fps proteins Encoded by Wild-type, Mutant and Revertant FSV DNAs 115 Tabel 4.2 Phosphoamino Acid Analysis of p1 3 0gag-fps and Total Cellular Protein 124 (ix) LIST OF FIGURES Page Figure 1.1 Amino Acid Sequences from the Kinase Domain of Twelve Oncogene Products and the Cyclic-AMP-Dependent Protein Kinase 13 Figure 3.1 Tryptic Phosphopeptide Analysis of FSV P i 4 0 g a g _ f p s 67 Figure 3.2 Cleavage of FSV P i 4 0 g a g - f p s with pl5 . . . . 73 Figure 3.3 Tryptic Peptide Analysis of pl5 Cleavage Fragments of FSV P140gaS- fP s 7 5 Figure 3.4 Cleavage of FSV P140ga€- fP s with V8 Protease. . . 78 Figure 3.5 Cleavage of [35S]methionine or 3 2 P-labelled FSV PMO^g-fps with V8 Protease 80 Figure 3.6 Tryptic Peptide Analysis of V8 Protease Cleavage Fragments of FSV P140Sag- fP s 83 Figure 3.7 Mapping V8 Protease Cleavage Fragments of FSV Pl 4 0gag-fps 85 Figure 3.8 Cleavage Sites for pl5 and V8 Protease on FSV P140 g a S- f P s 88 Figure 3.9 Tryptic Phosphopeptide Analysis of trFSV P140g a^- fP s , FSV P 1 3 0 g a g _ f p s and PRCII P105g a S- f p s 90 Figure 4.1 Synthetic Oligonucleotides Used to Mutate the Codon for Tyrosine-1073 of FSV PlSOgag-fps 100 Figure 4.2 Strategy for the Oligonucleotide-directed Mutagenesis of FSV 103 Figure 4.3 Partial DNA Sequencing of wtFSV, Mutant FSV-F(1073) and Revertant FSV-Y(1073) M13mplO Inserts . . . 105 Figure 4.4 Transformation of Rat-2 Cells Following Trans-fection with FSV DNAs 109 Figure 4.5 Analysis of Wild-type, Mutant and Revertant FSV-Transformed Rat-2 Cells for FSV PlSOg^-fps Synthesis and Tyrosine-specific Protein Kinase Activity . . . . . 113 (x) Page Figure 4.6 Tryptic Peptide Analysis of P 1 3 0 g a g - f p s Encoded by Wild-type, Mutant and Revertant FSVs 118 Figure 4.7 Phosphoamino Acid Analysis of P 1 3 0 g a g ~ f p s . . . 123 Figure 4.8 Whole Cell Phosphoamino Acid Analysis 127 Figure 5.1 The Synthetic Oligonucleotides Used to Mutate the Codon for Tyrosine-1073 of FSV P130 g a &- f p s . . 138 Figure 5.2 Partial DNA Sequencing of wtFSV and Mutant M13mpl0 FSV Inserts 139 Figure 5.3 Transformation of Rat-2 Cells Following Transfection with FSV DNAs 142 Figure 5.4 Expression of wt and Mutant P 1 3 0 g a g - f p s in Transformed Rat-2 Cells and Quantitation of Their Respective Tyrosine Protein Kinase Activities . . 145 Figure 5.5 Tryptic Phosphopeptide Analysis of wtFSV and Mutant FSV PlSOSag-^P8 Proteins 149 Figure 5.6 Tryptic Phosphopeptide Analysis of P I S O ^ - ^ 8 Encoded by Wild-type and Mutant FSVs 152 Figure 5.7 Phosphoamino Acid Analysis of wtFSV and Mutant FSV PlSOgag-fps Proteins 154 Figure 6.1 Comparison of the Structure of p-Fluorosulfonyl-benzoy1-51-Adenosine (FSBA) and Adenosine 5'-triphos-phate (ATP) 162 Figure 6.2 Inactivation of pl406 a&- fP s Kinase Activity By Treatment with FSBA 166 Figure 6.3 The Synthetic Oligonucleotdies Used to Mutate the Codon for Lysine-950 of FSV P130£ag-fPs . . 171 Figure 6.4 Morphological Phenotypes of Rat-2 Cells Following Transfection with FSV DNAs 175 (xi) Page Figure 6.5 Analysis of Wild-type and Mutant FSV Transfected Rat-2 Cells for FSV P130gag-fPs Synthesis and Tyrosine Protein Kinase Activity 178 Figure 6.6 Comparison of Tryptic Phosphopeptides From PlSOgaS-fps Encoded by Wild-type, Mutant FSV-S(1073) and Mutant FSV-R(950) 182 Figure 6.7 In Vitro Phosphorylation of FSV-R(950) P i 3 0 g a g ~ f p s by FSV P l 4 0 g a g - f p S 186 Figure 6.8 Tryptic Phosphopeptide Analysis of In Vitro Phosphorylated wtFSV P130£ ag- f p s and In Vitro Trans-phosphorylated FSV-R(950) PISOS^-^P8 . . . 189 (xii) LIST OF ABBREVIATIONS A adenine ADP adenosine 5'-diphosphate AEV Avian erythroblastosis virus AK adenylate kinase AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate APE alanine-proline-glutamic acid ASV Avian sarcoma virus bp base pair C cytosine cAPK cyclic-AMP dependent protein kinase CEFs chicken embryo fibroblasts cGPK cyclic-GMP dependent protein kinase CHO Chinese Hamster Ovary Ci Curie cpm counts per minute C-terminal carboxy-terminal portion of a protein dATP 2'-deoxyadenosine 5'-triphosphate DMEM Dulbecco's modified Eagle's medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid EDTA disodium ethylene diaminetetraacetic acid EGF epidermal growth factor ESV Esh sarcoma virus F phenylalanine FAV Fujinami associated virus FBS fetal bovine serum FSBA p-fluorosulfonylbenzoyl-5'-adensoine FSV Fujinami sarcoma virus G guanine G glycine x g times gravity A G° free energy (x i i i ) GA-FeSV Gardner-Arnstein feline sarcoma virus Gly glycine GMP guanosine 5'-monophosphate gs~ group specific antigen negative HAT hypoxanthine-aminopterin-thymidine IGF1 insulin-l ike growth factor-1 (somatomedin c) K lysine kb kilobase kbp kilobase pair kcal kilocalories kd kilodaltons Km Michaelis-Menton constant Lys lysine M Molar uCi micro-Curie ul microliter mCi milli-Curie mM millimolar NAD nicotinamide adenine dinucleotide NCP98 normal avian cellular fps gene product N-terminal amino-terminal portion of a protein PDGF platelet derived growth factor PCS Phase Combining System scintil lation cocktail PMSF phenylmethylsulfonylfluoride pTkl plasmid containing the Herpes Simplex-1 thymidine kinase gene PRC II Poultry Research Centre II sarcoma virus PRC IV Poultry Research Centre IV sarcoma virus R arginine Rf relative mobility RF replicative form RNA ribonucleic acid (xiv) RSV Rous sarcoma virus S serine SDS sodium dodecyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis Ser serine ST-FeSV Snyder-Theilen feline sarcoma virus T threonine T thymine TBE Tris-Borate-EDTA buffer TCA trichloroacetic acid TEMED N, N, N', N'-tetramethylethylenediamine Thr threonine TK thymidine kinase TK~ thymidine kinase minus mutant TLC thin layer cellulose TNE Tris-NaCl-EDTA buffer TPA phorbol tetradecanoate acetate TPCK L-(l-tosylamido-2-phenyl) ethyl chloromethyl ketone tr temperature resistant ts temperature sensitive Tyr tyrosine UR1 University of Rochester 1 sarcoma virus UR2 University of Rochester 2 sarcoma virus vol volume wt wild-type wt/vol weight by volume X an unspecified amino acid Y tyrosine Y73 Yamaguchi sarcoma virus ( X V ) ACKNOWLELXJEMENTS I would like to express my appreciation to my supervisory committee Drs. Julia Levy, George Spiegleraan and Ross MacGillivray, and to my research supervisor Dr. Tony Pawson for his support and constant enthusiasm during my research. In addition, I would like to thank the following people whose interest, support and assistance has contributed greatly to the successful completion of this thesis. A l l the past and present members of the Pawson group, especially Ehleen (knee-bone) for her friendship, encouragement and expert management of the laboratory, and Alasdair (Lord MacAuley) for taking-off numerous gels and lyophilizations and for alwaying being a good listener. The "Smith lab" for making mutagenesis possible and fun, and more specifically to Drs. Gary Pielak, David Goodin and Mike Smith for many enthusiastic, interesting, entertaining and informative discussions. Dr. Gerry Weeks for his advice, friendship, support and squash games. Gale Dawson who processed the words of this thesis with the expert care that only a sister could. Finally, I wish to express my deepest appreciation and sincerest thanks to Dennis for his constant encouragement, support, total friendship and love and especially his c r i t i ca l reading of this thesis. Financial support is gratefully acknowledged from the National Cancer Institute of Canada. (xvi) I would like to dedicate this thesis with love to my parents, Doris and David Weinmaster, whose constant love and support has sustained me throughout my student years. (xvii) - 1 -CHAPTER 1 1 . 0 INTRODUCTION 1.1 Classification of Retroviruses Retroviruses have been isolated from a wide variety of verte-brates, as well as invertebrates and have been grouped according to common morphological, biochemical and physical properties (for a com-plete review, see Teich, 1982). The Retroviridae virus family includes a l l viruses containing an RNA genome and an RNA-dependent DNA poly-merase (Fenner, 1975). The family is divided into three subfamilies: (1) Oncovirinae, including a l l the oncogeneic viruses and many closely related non-oncogenic viruses; (2) Lentivirinae, the "slow" viruses, such as visna virus; and (3) Spumavirinae, the "foamy" viruses that induce persistent infections without any cl inical disease. 1.2 Structure of Retroviruses The genomic RNA of a replication competent virus is a 60-70S dimer complex composed of two idential subunits, and which resembles eukaryotic rrRNA molecules in that there is a methylated cap structure at the 5' end and a polyadenylated tract at the 3' end (Teich, 1982). The genome can be used to direct protein synthesis in an in vitro translation system and therefore is considered to be of positive-sense. The genomes of a l l replication-competent retroviruses contain three - 9. -genes encoding the following viral proteins: (1) gag encodes the internal structural proteins found in the viral capsid; (2) pol codes for the RNA-dependant DNA polymerase or reverse transcriptase, and (3) env encodes the envelope proteins which are present in the v i ral membrane. The order of these three genes i s : 5'-gag-pol-env-3'. 1.3 Replication of Retroviruses Retroviruses encode and package the enzyme reverse tran-scriptase, which has both RNA-dependent and DNA-dependent DNA poly-merase activit ies. Both of these activities are essential to their mode of v iral replication which involves double-stranded DNA inter-mediates. Such intermediates can exist as unintegrated forms (linear or covalently closed circular molecules) or they may integrate into the cellular DNA of the infected host (proviral forms) (Varmus and Swanstrom, 1982). In the integrated state they can be transcribed like other cellular genes. Thus, a unique feature of retroviruses is that they may occur in nature as infectious elements (exogenous viruses) or as stably integrated proviruses within the cellular DNA of the host (endogenous viruses). The endogenous viruses are genetically transmitted as inherited genes from one generation to the next. These viruses may remain latent, they may be partially transcribed to produce viral mRNAs which are translated into virus-specified proteins, or they may become - 3 -activated to undergo a complete replication cycle with subsequent virus production, viremia and perhaps neoplasia (Teich, et a l . , 1982). On the other hand, the exogenous viruses are those which do not occur as integrated viral DNA copies until after infection. If this occurs in cells of the germ line, the exogenous virus may then become an endogenous virus and thus become a stable inheritable trait (Varmus and Swanstrom, 1982). 1.4 Oncogenic Retroviruses The oncogenic retroviruses, also known as RNA tumor viruses, can be divided into two classes based on their abil i ty to transform cel ls . One class rapidly causes neoplasias in animals due to the presence of an oncogene (onc+ viruses), while the other class of viruses induces malignancies by a complex mechanism which requires a long latent period (onc~ viruses) (Varmus, 1984). 1.4.1 Onc+ Oncogenic Retroviruses A number of the oncogenic retroviruses contain sequences which are essential for inducing tumors in animals and for causing morphological transformation of cells in culture. These sequences are termed one, for oncogene, since they are associated with the oncogenic - 4 -properties of the virus. In general, the one sequences are unrelated to sequences found in the genomes of replication competent viruses that lack transforming genes and they are not involved in viral replication. Both conditional and nonconditional mutations affecting the oncogenic potential of such viruses have been mapped to these sequences. Acqui-sition of viral transforming genes usually involves the loss of v iral genes required for replication. As a result, most of the viruses in this class are replication defective and can only be grown in the pre-sence of a helper virus; however, Rous sarcoma virus (RSV) is the exception to this rule (Bishop, 1983; Varmus, 1984). 1.4.1.1 Transduction of Cellular Oncogenes The origins of retroviral oncogenes were revealed when both avian (Stehelin, et a l . , 1976) and mammalian (Spector, et a l . , 1978) DNAs were shown to contain nucleotide sequences closely related to the oncogene of RSV. These findings suggested that a l l vertebrates possess a highly conserved gene that is related to a viral oncogene. As other retroviral oncogenes were discovered, each was shown to have a cellular homologue from which the viral oncogene was apparently derived. The homologous cellular genes, known as c-oncogenes (c-onc's) (Coffin, et a l . , 1981), are proto-oncogenes since they are the normal cellular progenitors of the viral oncogenes (v-onc's) and represent the targets for transduction by retroviruses (Varmus, 1984). - 5 -The c-onc's are evolutionarily well conserved; control of their expression appears to be modulated during growth and development which suggests that the c-onc encoded proteins may provide important physiological functions required for cellular growth and development (Bishop, 1983; Muller, et al., 1983; Varmus, 1984). It is generally considered that preexistent retroviruses incorporate c-oncogenes by recombination (Bishop, 1981). Retroviruses appear to be particularly adept at recombination: their genomes fre-quently exchange segments of variable size during mixed infections (Coffin, 1979) and their DNA forms enter the host genome as part of their normal replication cycle (Varmus, 1982a). Since proto-oncogenes and retroviral genes are unrelated, the transduction of c-onc sequences must be a complex and infrequent event because at least two illegitimate recombinations would be required to transduce a cellular sequence into a retroviral vector (Bishop, 1983; Duesberg, 1983; Varmus, 1984). Specific mutations are probably also necessary to convert a proto- oncogene (c-onc) into an oncogene (v-onc). 1.4.2 One" Oncogenic Retroviruses The second class of oncogenic viruses includes viruses that do not carry one sequences transduced from host-cellular genomes and do not appear to contain coding sequences for proteins other than those involved in viral replication. Nonetheless, the oncogenic viruses - 6 -lacking one sequences are capable of inducing a variety of neoplasms, but these tumors generally appear only after a long latent period; these viruses are also unable to transform cells in tissue culture (Teich, et al., 1982). Evidence suggests that tumors which arise following infection with v-onc~ retroviruses contain mutant cellular oncogenes that have been activated by proviral insertions (Varmus, 1982b). Integration of a provirus into the host genome is potentially mutagenic i f integration disrupts a vital region of the host genome (Bishop, 1983). Indeed, i t is generally thought that such insertion mutations are primary events in tumorgenesis and their effect is to stimulate expression of a cellular gene (c-onc) through the strong viral promoter or enhancer element present within the proviral long terminal repeat (LTR) (Hayward, et al., 1981; Neel, et al., 1981; Payne, et al., 1981; Payne, et al., 1982; Fung, et al., 1983; Cuypers, et al., 1984; Nusse, et al., 1984). 1.5 One and Proto-onc genes The retroviral oncogenes are transduced, multiply mutated, and highly tumorigenic forms of cellular proto-oncogenes (Varmus, 1984). Even though v-oncogenes appear to be copies of normal cellular genes, the nucleotide sequences of c-oncogenes and v-oncogenes has revealed many differences (Takeya and Hanafusa, 1983; Duesberg, 1983; Bishop, 1983; Huang, et al., 1985)). The v-oncogenes appear to be truncated versions of c-oncogenes that have acquired multiple point - 7 -mutations in addition to deletions of various sizes (Bishop, 1983; Duesberg, 1983). However, despite these differences the v^onc's demon-strate considerable homology with c-onc sequences (Karess, et a l . , 1979; Wang, et a l . , 1979; Wang, et a l . , 1980; Takeya, et a l . , 1982; Sodroski, et a l . , 1984). Although the protein products of the v i ral and cellular genes have extensive structural and functional homologies (Bishop, 1983; Varmus, 1984), significant differences exist, presumably due to the changes mentioned above. The viral proteins are usually expressed at a higher level and are sometimes truncated versions of the cellular proteins fused to other viral or cellular proteins (Duesberg, 1983). In addition, substitutions of single amino acids or blocks of amino acids may occur, and the sites and degree of phosphorylation may differ (Bishop and Varmus, 1982; Duesberg, 1983). These changes may be responsible for the functional differences which have been detected between viral oncogenic proteins and their normal cellular counter-parts. 1.6 Retroviral Oncogene Products Extensive genetic evidence indicates that the protein product of a single RNA tumor virus gene is generally responsible for the malignant transformation of virus infected cells (Bishop, 1983; Duesberg, 1983; Varmus, 1984). Over the past few years the protein - 8 -products of a number of different oncogenes have been identified (Table 1.1). Viral oncogenes may be functionally grouped into two broad classes: (1) those that encode or are likely to encode a tyrosine specific protein kinase activity and (2) those that do not encode this enzymatic activity. 1.6.1 Tyrosine Kinase-Negative Class of Oncogene Proteins The tyrosine protein kinase-negative class of oncogenes can be subdivided into a number of functional subclasses. They consist of: (1) the v-sis oncogene of Simian sarcoma virus, which encodes the B chain of platelet-derived growth factor (PDGF) (Waterfield, et a l . , 1983; Doolittle, et a l . , 1983; Johnsson, et a l . , 1984); (2) the v-fos oncogene of FBJ sarcoma virus, v-myc of MC29 virus and v-myb of Avian myloblastosis virus which encode proteins that are found in the ce l l nucleus and the latter two oncogene proteins have been shown to bind DNA (Abrams, et a l . , 1982; Dormer, et a l . , 1982; Hann, et a l . , 1983; 1983; Al i talo, et a l . , 1983; Curran, et a l . , 1984; Eisenman, et a l . , 1985) and (3) the H-ras and K-ras oncogenes of murine sarcoma viruses, which encode guanosine nucleotide-binding proteins (Papageorge, et a l . , 1982). - 9 -TABLE 1.1 Retroviral Oncogenes Oncogene Viral Origin Viral Gene Product Cellular Horaologue Activity src Rous sarcoma virus v-src p60 p60C_SrC Tyrosine protein kinase yes Y73 avian sarcoma virus P90Eag"yeS Tyrosine protein kinase fgr Gardner-Rasheed feline sarcoma virus P70ga8_fgr Tyrosine protein kinase fps Fujinami sar-coma virus pl40gag-fps p98C-fpS Tyrosine protein kinase abl Abelson murine leukemia virus P120Wrabl , c-abl pi 50 Tyrosine protein kinase ros UR2 avian sarcoma virus p^ gag-ros Tyrosine protein kinase fes Snyder-Theilen feline sarcoma virus P85gag_feS p92 Tyrosine protein kinase erb-B Avian erythro-blastosis virus erb-B gp65 Truncated EGF recep-tor Potential tyro-sine protein kinase fms McDonough feline sarcoma virus Potential tyro-sine protein kinase mil MH2 avian virus pl00gag^nil Potential serine/ threonine protein kinase raf 3611 murine sarcoma virus Potential serine/ threonine protein kinase mos Moloney murine sarcoma virus mos p37 Potential protein kinase sis Simian sarcoma virus sis p28 PDGF B-chain PDGF agonist Ha-ras Harvey marine sarcoma virus „ v-H-ras p21 GTP binding Kl-ras Kirsten marine sarcoma virus „.v-K-ras P21 ^jC-K-ras GTP binding foe FBJ murine osteosarcoma virus p55fOS Possible DNA bind-ing protein myc Avian myelc— cytomatosis virus K29 pll0gag-myc Binds DNA myb Avian myelo-blastosis virus p480Vb Possible DNA bind-ing protein The table l i s ts a number of oncogenes, a virus strain which contains the oncogene, the viral gene product encoded by the oncogene and i t s known or proposed activity and where identified the cellular homologue. The information in the table was obtained from Heldin and Westermark (1984) and various references sited in the text. - 10 -1.6.2 Tyrosine Protein Kinase-Positive and Kinase-Related Class of  Oncogene Proteins The oncogene products in this class a l l share structural and/or functional homology with the transforming protein of RSV and are collectively known as the src family of kinases. This family i s encoded by a number of independently isolated, acutely transforming retroviruses (Table 1.1). Each of these viruses carries one of six transduced cellular genes, a l l of which apparently originated from loci different from src. These oncogenes include yes, fgr, fps, fes, abl and ros; a l l of which encode proteins that have detectable tyrosine kinase activity. These viral oncogenes have been molecularly cloned and hybridization studies have revealed that the region of strongest homology corresponds to the kinase domain. These homologous regions are in the 3' ends of src, yes, fes, fps, and fgr, the 5' end of abl and the middle of ros (Takeya and Hanafusa, 1983; Kitamura, et a l . , 1982; Shibuya and Hanafusa, 1982; Hampe, et a l . , 1982; Naharro, et a l . , 1984; Reddy, et a l . , 1983; Neckameyer and Wang, 1984). The correspond-ing amino acid sequences deduced for the proteins encoded by these genes are a l l similar to the carboxy-terminal domain of p 6 0 s r c that carries the kinase activity (Levinson, et a l . , 1981; Brugge and Darrow, 1984). Using the amino acid sequence of p 6 0 s r c as a reference point (Schwartz, et a l . , 1983), the computed homologies are - l i -as follows: yes 90% (Kitamura, et a l . , 1982); fps 48% (Shibuya and Hanafusa, 1982); abl 51% (Reddy, et a l . , 1983); fgr 82% (Naharro, et a l . , 1984) and fes 45% (Hampe, et a l . , 1982). The regions of these oncogene proteins that contain the major phosphotyrosine acceptor site show either identical or conservatively altered amino acid sequences (Figure 1.1), a l l of which result in predicted secondary structures that are identical (Mark and Rapp, 1984). This area also contains the highly conserved ala-pro-glu (APE) sequence which is thought to specify a functional domain that is essential for the tyrosine protein kinase and cellular transforming activities of p 6 0 s r c (Byrant and Parsons, 1984). These comparisons indicate that the genes encoding tyrosine protein kinase activity comprise a single family of genes (Figure 1.1). In fact a l l protein kinases, irrespective of amino acid specificity, may be encoded by a single family of genes that have diverged from a common origin. This was f i rs t reported by Barker and Dayhoff (1982) who used a computer search program to show that the cyclic-AMP dependent protein kinase (cAPK), the only serine protein kinase whose complete sequence was known (Shoji, et a l . , 1981), i s related to p 6 0 s r c . The analysis revealed that residues 259 to 485 of p 6 0 s r c have 22% sequence identity with residues 38-258 of the catalytic subunit of cAPK. Alignment of the two sequences to obtain maximal amino acid identity aligns lysine-295 in p 6 0 s r c with lysine-71 in the catalytic subunit. Both of these conserved lysine - 1 2 -Figure 1 . 1 Amino acid sequences from within the protein-kinase domain of 1 2 oncogene products and the cyclic-AMP-dependent protein kinase (cAPK) are arranged to show their similarity. A stretch in the middle of each sequence has been omitted; the number of subunits omitted is indicated. Each dot represents a one-amino-acld gap introduced to attain the best alignment. The amino acids are represented by their one-letter codes: A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glut amine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; Y, Tyrosine. The sequences blocked are discussed in the text. References for the sequence data are cited in the text. s r c KL GQGCFG EVWMGTW'JDTTR , . . V A I K TLKP — 83 y e s KL GQGCFG EVWMGTWNGTTK VA[ K TLKL — 83 f g r RL GTGCFG DVWLGMWNGSTK K TLKP — 83 a b l Kl GGGQYG EVYEGVWKKYSLT . , , VAV K TLKE — 84 fps RI GRGNFG EVFSGRLRADNTP . . , ,VAV K SCRE — 85 f e s 01 GRGNFG EVFSGRLRADNTL K SCRE — 85 r o s LL GSGAFG EVYEGTALDILADGSGESRVAV K TLKR — 91 e r b - B VI GSGAFG TI YKGLWIPEGEK. . V T I P V A I K ELRE — 84 fms TI GTGAFG KVVEATAFGLGKED . A V L K V A V K MLKS - 155 mi 1 RI GSGSFG TVYKGKWHGD . . , ,VAV K ILKV — 85 r a f RI GSGSFG TVYKGKWHGD VAV K ILKV — 85 mos RL GSGGFG SVYKATYHG K QVNK - 100 CAPK TL GTGSFG RVMLVKHMETGNH YAM K ILDK — 86 VHRDLRAANILVGENL .. VCKVADFGLARLIEDNE Y TARQGAKF..PIKWT APE A IHRDLRAANILVGDNl ...VCKIADFGLARLIEDNE Y TARQGAKF..PIAWT APE A IHROLRAANILVGERL ,, VCK[ADFGLARLIEDNE Y NPRQGAKF..PIKWT APE A IHRDLAARNCLVGENH , LVKVAOFGLSRLMTGDT Y TAHAGAKF..PIKWT APE S IHRDLAARNCLVTEKN ...TLKISOFGMSRQEEOGV Y ASTGGMKQI.PVKWT APE A 1 IHRDLAARNCLVTEKN . . . VLKISDFGMSREEADGV Y AASGGLRLV.PVKWT APE A CO IHROLAARNCLVSEKQYGSCSRVVKIGDFGLARDIYKND Y YRKRGEGLL.PVRWM APE S 1 VHRDLAARNVLVKTPQ , K EYHAEGGKV.PIKWM ALE S IliRDVAARNVLLTSGR VAKIGDFGLARDIMNDS N YIVKGNARL.PVKWH APE s IHRDMKSNNIFLHEGL , E SQQVEQPTG.SILWM APE V IHRDMKSNNIFLHEGL, . G SQQVEQPTG.SVLWM APE V G RQASPPHIGGTYTHQ APE I IYRDLKPENLLIDQQG, . W T.LCGT PE.YL APE I - 14 -residues have been shewn to react specifically with the ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine (FSBA) (Zoller, et a l . , 1981 Kamps, et a l . , 1984). FSBA also reacts with the homologous lysine residue in the cyclic-GMP dependent protein kinase (cGPK), which has 42% sequence homology with cAPK within the catalytic region (Hashimoto, et a l . , 1982). Thus, the three-dimensional structure of the ATP-binding regions of both the cyclic nucleotide dependent serine kinases and p 6 0 s r c tyrosine kinase probably orient the homologous lysine residue in a similar conformation. In addition to this highly con-served lysine residue, these proteins a l l contain a linear array of glycines that have been proposed to function in nucleotide binding (Adams, et a l . , 1973; Rossmann, et a l . , 1974; Pai , et a l . , 1977; Wierenga and Hoi, 1983). Together, these data provide convincing evidence that the sequence homology between the tyrosine protein kinases and the serine protein kinases reflect structural and func-tional homology, and indicate that the protein kinases, irrespective of their amino acid substrate specificity, share a common ancestry. In addition to the oncogenes discussed above, there are five viral transforming genes whose protein sequences show lesser and vary-ing degrees of homology to p 6 0 s r c and are therefore considered to be only related to the src family of tyrosine kinases. These related proteins are encoded by the oncogenes fms (Hampe, et a l . , 1984), mil (Jansen, et a l , 1984), raf (Mark and Rapp, 1984), mos (Van Beveren, - 15 -et a l , 1981) and erb-B (Privalsky, et a l . , 1983; and Yamamoto, et a l . , 1983). The existing homologies include the cluster of glycines and the conserved lysine contained within the putative ATP binding site, as well as the conserved APE sequence discussed above (Figure 1.1). However, the homologous phosphotyrosine acceptor site is not conserved and, except for erb-B (Gilmore, et a l . , 1985) and fms (Barbacid and Lauver, 1981) there is no evidence to date that these proteins possess tyrosine protein kinase activity. Recently, the protein products of the raf and mil oncogenes have been reported to be associated with a serine and threonine protein kinase activity (Moelling, et a l . , 1984). In addition, a serine-threonine protein kinase has been reported to be associated with one mos-containing viral protein (Kloetzer, et a l . , 1983). It is interesting that site-specific mutagenesis of lysine-121 in mos, which is homologous to lysine-295 in p 6 0 s r c , destroys the transforming activity of mos (M. Hannick and D.J. Donoghue, personal communication). The meaning of this kinship is not yet fully understood. It i s likely that those transforming proteins related to the src family of oncogenic tyrosine kinases also possess some of the functions of protein kinases. On the other hand, a phylogenetic tree of the src family of oncogenes, constructed on the basis of the relatedness of their conserved sequences (Mark and Rapp, 1984), corroborates the existing data and indicates that the src family of oncogenes has indeed evolved from a common ancestor. - 16 -1.7 Cellular Tyrosine Protein Kinases The diversity of tyrosine kinase encoding genes captured by retroviruses suggests that the vertebrate genome codes for a number of such proteins. In fact the f i rs t tyrosine kinase identified in normal cells was the cellular homologue of p 6 0 s r c (Collett, et a l . , 1978; Oppermann, et a l . , 1979; Col lett , et a l . , 1979b). Since then a number of normal cellular homologues of the oncogenic tyrosine kinases have been identified. Their differential expression in various cel l types suggests that tyrosine phosphorylation plays a role in normal cellular growth and differentiation (Bishop, 1983). Although transformation was previously thought to result from the introduction of a totally foreign activity into the ce l l , the discovery of normal cellular tyrosine kinases suggested that either over-expression of a normal enzyme or a subtly altered version of that enzyme was responsible for the cellular t ransformation. The idea that tyrosine kinases may be involved in the regula-tion of normal cellular growth is substantiated by the fact that the receptors for epidermal growth factor (EGF) (Cohen, et a l . , 1980; Ushiro and Cohen, 1980; Buhrow, et a l . , 1982), platelet-derived growth factor (PDGF) (Ek, et a l . , 1982; Heldin, et a l . , 1983), insulin (Petruzzelli, et a l . , 1982; Roth and Cassell, 1983) and somatomedin C (IGF1) (Jacobs, et a l . , 1983a; Rubin, et a l . , 1983) a l l possess tyro-sine specific protein kinase activity. The binding of the respective - 17 -growth factors to their receptors results not only in the phosphoryla-tion of the receptor, but also in the phosphorylation of cellular substrates at tyrosine residues. This suggests that tyrosine phos-phorylation is involved in the transmission of the mitogenic signal (Cooper, et a l . , 1984b). In addition to the cellular tyrosine kinases that are stimu-lated by growth factors, a number of other tyrosine kinases in normal cells have been described recently. It has been possible to detect these kinases by using synthetic peptide substrates containing tyro-sine, but lacking serine and threonine (Casnellie, et a l . , 1982; Wong and Goldberg, 1983a; Dasgupta and Garbers, 1983; Swarup, et a l . , 1983). These cellular tyrosine kinases are unrelated to the oncogenic tyrosine protein kinases and are not known to be stimulated by growth factors. For example, a 53,000 to 56,000 dalton tyrosine kinase has been detected in the LSTRA murine lymphoma cel l line and in normal T lymphocytes (Swarup, et a l . , 1983; Voronova, et a l . , 1984). Also, a tyrosine protein kinase activity of 75,000 daltons (p75) has been described in the soluble and microsomal fraction of rat liver (Wong and Goldberg, 1983a). Tyrosine protein kinases have also been detected indirectly by the presence of phosphotyrosine in developing embryos (Eisenman and Kinsey, 1982), the phosphorylation of tyrosine residues in Band 3 of erythrocyte membranes (Dekowski, e t a l . , 1983) and the - 18 -phosphorylation of synthetic peptides by extracts of various rat tissues and developing embryos (Swarup, et a l . , 1983; Dasgupta and Garbers, 1983). In total, the normal eukaryotic genome may encode as many as twelve different protein kinases that phosphorylate themselves and other substrates at tyrosine residues. 1.8 Cellular Substrates of Tyrosine Kinases The activity of virally-coded tyrosine protein kinases accom-panies and is necessary for the malignant transformation by these viruses. Transformation of cells by retroviruses encoding tyrosine kinases results in a five to ten fold increase in cellular phospho-tyrosine levels (Cooper and Hunter, 1981a), which indicates that the phosphorylation of cellular target proteins may be involved in the mechanism of transformation. Inappropriate phosphorylation of key regulatory proteins could disrupt normal cellular growth control mechanisms. The detection of tyrosine kinase activity associated with certain growth factor receptors and the enhancement of cellular phos-photyrosine following treatment of cells with these growth factors (Hunter and Cooper, 1981; Erikson, et a l . , 1981; Cooper, et a l . , 1982) suggest a link between tyrosine phosphorylation and cel l growth. It may be that some oncogenic proteins have the same activity and over-lapping protein specificity as some growth factor receptors, but are no longer subject to the same regulation. - 19 -The link between growth factors and transformation has been further strengthened by the recent findings that the v-sis oncogene of Simian sarcoma virus is related to the gene encoding PDGF (Doolittle, et a l . , 1983; Waterfield, et a l . , 1983) and that the transforming gene of Avian erythroblastosis virus, (erb-B) is apparently a truncated version of the EGF receptor (Downward, et a l . , 1984a; Ullr ich, et a l . , 1984). Both of these findings suggest mechanisms whereby oncogene products could subvert normal growth control mechanisms and lead to the malignant state. The identification and functional characterization of sub-strate proteins for tyrosine kinases is obviously important, since they may be involved in the control of cellular growth in both normal and malignant cel ls . The cellular proteins phosphorylated at tyrosine in cells transformed by the members of the src family of viruses are similar to each other (Cooper and Hunter, 1981b). This is not surpris-ing considering the proposed common ancestry of the src family cata-ly t ic domains, as discussed in section 1.6.2 . It also reinforces the idea that these viruses probably transform cells by a common mechanism involving tyrosine phosphorylation. Malignant transformation by these RNA tumor viruses is a multifaceted process which alters the cells in a number of ways, including loss of contact inhibition, abnormal glucose metabolism and alterations in cel l shape (Bishop and Varmus, 1982; Cooper and Hunter, - 20 -1983b). A number of cellular targets have been identified by virtue of the fact that they may be directly involved in the above mentioned cellular changes. For example, the change in cel l shape i s , in some cases, correlated with an enhanced phosphorylation of the cytoskeleton protein vinculin, a protein found specifically localized in adhesion plaques (Geiger, 1979; Rohrschneider, 1980; Shriver and Rohrschneider, 1981). It has been proposed that vinculin acts as a linker between the plasma membrane and the termini of actin-containing microfilament bundles (Burridge and Feramisco, 1980) and would therefore be important in maintaining cel l shape. Since p 6 0 s r c i s localized in the adhesion plaques in RSV transformed cells (Rohrschneider, 1980; Sefton, et a l . , 1981; Shriver and Rohrschneider, 1981; Nigg, et a l . , 1982), phosphorylation of vinculin by p 6 0 s r c may interfere with i ts function and result in disorganization of the actin-containing micro-filaments leading to an alteration in cel l shape. However, several findings indicate that vinculin phosphorylation alone is not sufficient to induce the morphological changes seen upon transformation. Most important, alterations in ce l l shape and phosphorylation of vinculin are not always correlated (Rohrschneider and Rosok, 1983), and only 1.0% of the vinculin in the transformed cel l is phosphorylated (Sefton, et a l . , 1981). In addition, quantitative changes in both fibronectin and vinculin may contribute to the changes in cel l shape (Olden and Yamada, 1977; Iwashita, et a l . , 1983). - 21 -A well known characteristic of tumor cells is an increased rate of aerobic glycolysis,, known as the Warburg effect (Racker, 1972). The glycolytic enzymes enolase, phosphoglycerol mutase and lactate dehydrogenase have been shown to contain phosphotyrosine (Cooper, ejt a l . , 1983a). However, l ike vinculin, only a small fraction of these enzyme molecules are phosphorylated, which raises questions as to whether the degree of phosphorylation could produce the enhanced rate of glycolysis observed in transformed cel ls . In fact, of the eleven enzymes involved in the breakdown of glucose, the three glycolytic enzymes phosphorylated at tyrosine do not catalyze rate limiting steps, and the key enzyme phosphofructokinase does not appear to be phosphory-lated at tyrosine (Cooper, et a l . , 1983b; Cooper, et a l , 1984a) Perhaps the enhanced rate of glycolysis seen with transformed cells is the result of an increased rate in glucose transport by such malignant cells (Bissell , et a l . , 1973). The level of phosphotyrosine in a normal cel l is less than 0.1% of the total acid-stable phosphoamino acids (Sefton, et a l . , 1980). Therefore, cellular substrates for tyrosine kinases should be detected by virtue of their enhanced phosphorylation at tyrosine res i -dues following infection with viruses encoding tyrosine kinase activity, or treatment of cells with growth factors that activate receptor tyrosine kinases. This approach has revealed a number of tyrosine protein kinase substrates, but the identity and function of many of these proteins is unknown. - 22 -A 36,000 dalton protein (p36 or 36K) is the most extensively characterized tyrosine kinase substrate, being the f i rst cellular target identified (Radke and Martin, 1979) and an abundant cellular protein of chick fibroblasts (Radke, et a l . , 1980; Cooper and Hunter, 1983a). p36 is phosphorylated at tyrosine in cells transformed by oncogenic viruses that encode tyrosine protein kinase activity and only in some cells following exposure to growth factors (Cooper and Hunter, 1983b). The 36K phosphoprotein co-immunoprecipitates with gpggerb-B f r o m ^ transformed cells (Gilmore, et a l . , 1985) and with p 6 0 s r c from RSV transformed cells (Dehazya and Martin, 1985), suggesting that these complexes may represent stable enzyme-substrate associations. However, p36 is not extensively phosphorylated under the following conditions: resting or growing normal cells; infection with leukosis viruses; treatment with tumor promoters; and transformation by chemicals, DNA viruses or retroviruses that do not encode tyrosine pro-tein kinases (Cooper and Hunter, 1983b). The phosphorylation of p36 is not always correlated with conditions of mitogenic stimulation (Cooper, et a l . , 1982; Decker, 1982; Nakamura, et a l . , 1983) and morphological changes seen with transformation do not always accompany p36 phosphory-lation (Nakamura and Weber, 1982; Cooper, et a l . , 1983b). Both the phosphorylated and unphosphorylated forms of p36 are found predomin-ately in the plasma membrane, where i t may perform a structural func-tion (Cooper and Hunter, 1982; Courtneidge, et a l . , 1983; Greenberg and Eldeman, 1983; Radke, et a l . , 1983). Even though p36 is abundant in fibroblasts, i t i s not found in a l l cells (Cooper and Hunter, 1983b). - 23 -A 42,000 dalton phosphoprotein is detected in chick cel ls , but not in mammalian cells transformed by the avian sarcoma viruses (Cooper and Hunter, 1981a). Treatment of density-inhibited chick cells with a wide variety of mitogenic agents, EGF, PDGF, trypsin and the phorbol ester TPA, stimulates the immediate phosphorylation of two 42,000-dalton cellular proteins at tyrosine (Bishop, et a l . , 1983; Gilmore and Martin, 1983; Nakamura, et a l . , 1983; Cooper, et a l . , 1984b), which indicates that p42 may be involved in the regulation of ce l l division. Unlike that found in RSV transformed chick cel ls , treatment of chick cells with mitogenic agents results in very few phosphorylated proteins, although p42 is by far the most prominent of these few (Nakamura, et a l , 1983). Phosphorylation of p42 may be involved in the delivery of the mitogenic signal, but as yet i t s identity and function remain an enigma. Along with the 36K and 42K proteins, an 81,000 dalton protein (p81) is also phosphorylated at tyrosine following treatment of A431 cells with EGF (Hunter and Cooper, 1981c). The same protein is also found phosphorylated in 3T3 cells transformed by ST-FeSV; however, i t i s not phosphorylated in other types of transformed cells or in normal cel ls . The physiological significance of this EGF-induced phosphoryla-tion is questionable, since the EGF dose required to obtain phosphory-lation of pSl leads to an inhibition of the growth of A431 cells (Gi l l and Lazar, 1981). In fact, in fibroblasts responsive to mitogenic stimulation by EGF, neither p36 nor pSl is phosphorylated following treatment with EGF (Cooper, et a l . , 1984b). - 24 -In RSV transformed cel ls , p60 f a r c i s found in a complex with a 50,000 dalton protein (p50) and a major heat shock protein (p89) (Sefton, et a l . , 1978; Hunter and Sefton, 1980; Brugge, et a l . , 1981; Oppermann, et a l . , 1981). The oncogenic tyrosine kinases p l 0 5 gag- fps (Adkins, et a l . , 1982), m)^^yes (Lipsich, et a l . , 1982) and P140 g ag~ fP s (Pawson, et a l . , 1981) are also found in a complex with p50 and p89. It is thought that the complex is involved in shuttling p 6 0 s r c from i ts site of synthesis on soluble polysomes to the plasma membrane. p50 is phosphorylated at a single tyrosine residue (Hunter and Sefton, 1980; Oppermann, et a l . , 1981) in transformed cel ls , but not in normal cells (Brugge and Darrow, 1982; Gilmore, et a l . , 1982). Whether the tyrosine phosphorylation of p50 is fortuitous or functional, i t seems likley that p50 is a substrate for the tyrosine protein kinase with which i t is associated. Despite the identification of several cellular proteins con-taining high levels of phosphotyrosine in viral ly transformed cells and cells treated with growth factors, there is no direct proof that the phosphorylation of these proteins is necessary or sufficient for trans-formation or growth stimulation. If tyrosine phosphorylation is involved in these processes, then phosphorylation of specific proteins must change their function in a manner that contributes to the altered cellular phenotype. However, i t has never been shown that tyrosine phosphorylation affects the function of a substrate in a meaningful way. It may be that the physiologically significant substrates for - 25 -retroviral kinases are low abundance proteins which have not yet been identified, or that the phosphorylation of substrates other than proteins is crucial for transformation. 1.8.1 Indirect Cellular Substrates of Tyrosine Kinases Although much attention has been focused on tyrosine phos-phorylation, analysis of specific proteins, such as the ribosomal protein S6, reveals that protein phosphorylation at serine residues is also quantitatively altered by the expression of transforming proteins (Decker, 1981; Blenis and Erikson, 1984; Blenis, et a l . , 1984; Mailer, et a l . , 1985) and treatment of cells with mitogenic agents (Nilsen-Hamilton, et a l . , 1982, Martin-Perez and Thomas, 1983; Chambard, et a l . , 1983; Trevillyan, et a l . , 1984; Martin-Perez, et a l . , 1984; Novak-Hofer and Thomas, 1984; Blenis, et a l . , 1984). The wide spread correlation between S6 phosphorylation and the growth-promoting actions of a diverse group of agents suggests that S6 serine phosphorylation plays an important role in growth regulation. The fact that oncogenic tyrosine kinases and growth promoting agents associated with tyrosine specific protein kinases enhance S6 phosphorylation on serine residues implies that such kinases are capable of regulating the enzymes that control S6 phosphorylation. Recently, some oncogenic tyrosine kinases have been reported to show lipid-phosphorylating activity (Sugimoto, e t a l . , 1984; Macara, - 26 -et a l . , 1984), which implicates these kinases in phosphatidylinositol turnover and the role this turnover plays in the activation of protein kinase C (Marx, 1984; Berridge, 1984). Although there is a certain degree of skepticism regarding the purity of the oncogenic tyrosine kinases used in these studies, they remind us of the possibility that the transforming proteins of these viruses may have some other capacity as cr i t ica l for transformation as protein phosphorylation. The possi-b i l i t y remains that the src family of transforming proteins may ac t i -vate pathways leading to the malignant phenotype through the generation of second messengers, and not by the covalent modification of various substrate proteins. 1.9 Characteristics of Tyrosine Protein Kinases Tyrosine phosphorylation is a recently recognized protein modification f i rs t identified in a number of v iral transforming pro-teins (Eckhart, et a l . , 1979; Collett, et a l . , 1979a; Hunter and Sefton, 1980; and Witte, et al •, 1980). Although i t has been assumed that the phosphotransferase activity is intrinsic to these transforming proteins, only src (Gilmer and Erikson, 1981; McGrath and Levinson, 1982), abl (Wang, et a l . , 1982) and fps (J. Stone, personal communica-tion) have been molecularly cloned in E.coli and the expressed proteins shown to possess tyrosine protein kinase activity. In contrast, the kinase activity associated with middle T antigen of polyoma virus (Eckhart, e t a l . , 1979) has since been shown to be the property of p60c s r c , which binds specifically to middle T antigen (Courtneidge and Smith, 1983). Given the close familial relationship between the oncogenic tyrosine protein kinases, i t i s not surprising that these kinases share biochemical properties as well. Because tyrosine protein kinases form a class functionally distinct from other protein kinases, the Nomen-clature Commission of the International Union of Biochemistry has assigned them a separate number E.C.2.7.1.37 (Recommended name: Protein-tyrosine kinase. Reaction: ATP + protein tyrosine = ADP + protein O-phosphotyrosine). A l l of the tyrosine specific kinases characterized to date autophosphorylate at tyrosine residues and have activity independent of cyclic nucleotides or Ca (Bishop and Varmus, 1982). Many but not a l l of the enzymes show a preference for 2+ 2+ Mn over Mg^  as a cof actor, but this may reflect inhibition 2+ of endogenous phosphatases by Mn . Although ATP is the preferred phosphate donor, dATP and GTP can be substituted in some cases (Richert, et a l . , 1982). Interestingly, the enzymatic properties of the UR2 transforming protein, pesgag-1"08, are distinctive from those of the other avian sarcoma virus protein kinases in cation pre-ference, pH optimum, and phosphate donors (Feldman, et a l . , 1982). Studies using synthetic peptides as substrates have shown that the tyrosine kinases have a tight specificity for tyrosine as the acceptor amino acid and they do not phosphorylate serine, threonine or - 28 -hydroxyproline (Pike, et a l . , 1982; Hunter, 1982; Wong and Goldberg, 1983b). The specificities of the cyclic nucleotide dependent serine and threonine protein kinases have also been thoroughly explored by the use of synthetic peptides (Zetterquist, et a l , 1976; Kemp, et a l . , 1976; Kemp, et a l . , 1977; Kemp, 1979; Glass and Krebs, 1982). These studies and others indicate that the canonical recognition sequence for these kinases contains charged residues near the target amino acid. However, sequencing of tyrosine kinase recongition sites (Smart, et a l . , 1981; Neil , et a l . , 1981; Patschinsky, et a l . , 1982; Cooper, et a l . , 1984a) and studies with synthetic peptides (Hunter, 1982) demonstrate that while acidic residues are a factor in substrate recognition, the presence of acidic residues in the primary sequence near the target tyrosine residue is not an absolute requirement for phosphorylation by tyrosine protein kinases (Gallis, et a l . , 1983; Guild, et a l . , 1983; Cooper, et a l . , 1984a). It seems probable that secondary structure is also important for the recognition of phosphory-lation sites by these enzymes. The protein bound tyrosine phosphate i s a high energy l ink-age. The energy of hydrolysis (AG°) of protein bound tyrosine phos-phate has been reported to be -9.48kcal (assuming an approximate &G° of -10 kcal for hydrolysis of ATP) (Fukami and Lipmann, 1983). This is important since hydrolysis or formation of such a high energy tyrosine phosphate bond in proteins could conceivably bring about a conforma-tional change in the protein resulting in an altered functional state - 29 -of the molecule. In support of this, enhanced tyrosine phosphorylation of the insulin receptor kinase and the src kinase has been reported to stimulate the tyrosine kinase activity intrinsic to these proteins (Rosen, et a l . , 1983; Purchio, et a l . , 1983; Collett et a l . , 1984, Brown and Gordon, 1984). 1.10 Phosphorylation of Tyrosine Kinases - A Possible Role in  Regulating Activity 1.10.1 Tyrosine Phosphorylation and the Regulation of Kinase Activity Like the phosphorylation of serine and threonine residues (Krebs and Beavo, 1979), the phosphorylation of tyrosine residues is a reversible event (Fukami and Lipmann, 1983). The dephosphorylation of phosphotyrosine residues is carried out by phosphotyrosine-specific protein phosphatases (Foulkes, 1983), which can be distinguished from phosphoserine or phosphothreonine-specific phosphatases by the use of orthovanadate (Swarup, et al•, 1982). Treatment of RSV transformed cells with vanadate has revealed hyperphosphorylated forms of p 6 0 s r c . (Collett, et a l . , 1984; Brown and Gordon, 1984). This enhanced phosphorylation was due to an increase in tyrosine rather than serine phosphorylation of p 6 0 s r c , and could be correlated with a significant increase in i ts tyrosine kinase activity. The increase in tyrosine phosphorylation of - 30 -rj60br^ was due to the appearance of newly characterized sites of tyrosine phosphorylation in the amino-terminal portion of the molecule. Similar structurally and functionally modified forms of the RSV src protein have also been detected by lysis of transformed cells in the presence of ATP-Mg2* (Purchio, et a l . , 1983; Collett, et a l . , 1983). Only when lysis was conducted at high ATP concentrations were the new sites phosphorylated. Perhaps this phosphorylation when suf f i -ciently extensive exerts an allosteric effect on p 6 0 s r c resulting in a net increase in the protein kinase activity of the enzyme. These data introduce the possibility that within transformed cells there may be transient, rapidly modified molecules of p 6 0 s r c that are only detected under conditions which inhibit phosphotyrosine specific phos-phatases. Alternatively, vanadate may directly or indirectly activate other kinases which results in the phosphorylation of these recently identified phosphotyrosine sites in p 6 0 s r c . The vanadate experiments appear to suggest that the level of phosphorylation of tyrosine protein kinases and their cellular sub-strates w i l l depend upon the phosphotyrosine specific phosphatases in the ce l l . Since reversible phosphorylation is thought to be a major mechanism for regulating protein function, i t seems reasonable to con-clude that the phosphorylation of tyrosine protein kinases represents a regulatory mechanism. Additional support for this possibility comes from the observation that incubation of the insulin receptor under conditions in which i t autophosphorylates at tyrosine residues - 31 -stimulates i t s ability to catalyze the phosphorylation of exogenous substrates at tyrosine residues (Rosen, et a l . , 1983). The phosphory-lated receptor remained active after the removal of insulin; however, dephosphorylation of the phosphorylated receptor rendered i t dependent upon insulin for optimal activity. These results suggest that both the dissociation of bound insulin and dephosphorylation of the receptor may be required to terminate the insulin signal. This study with the insulin receptor and those mentioned above for the src kinase suggest a possible role for tyrosine phosphorylation in the regulation of tyro-sine specific protein kinases and this thesis w i l l deal with this aspect in greater detail in the following chapters. 1.10.2 Serine and Threonine Phosphorylation and the Regulation of  Kinase Activity When studied in intact cel ls, the oncogene derived tyrosine kinases (Bishop, 1983; and Cooper and Hunter, 1983b), as well as the receptors for EGF (Downward, et a l . , 1984b, Cochet, et a l . , 1984), PDGF (Heldin and Westermark, 1984), insulin (Kasuga, et a l . , 1982) and IGFl (Jacobs, et a l . , 1983b), were a l l found to contain phosphoserine, and in some cases phosphothreonine, in addition to phosphotyrosine. In most cases the kinases involved in the phosphorylation of serine and threonine are unknown, as are the effects these phosphorylations have on the activity of the various tyrosine kinases. - 32 -The serine phosphorylation of p 6 0 s r c may be the product of the cyclic-AMP dependent protein kinase (cAPK) (Collett, et a l . , 1979a), however other kinases are probably involved. A mutant of RSV which lacks the major site of serine phosphorylation in p 6 0 s r c i s unaltered in both i ts biochemical and biological activities (Cross and Hanafusa, 1983), suggesting that this site of phosphorylation is not essential for the activity of the protein. However, phosphorylation by cAPK increases the abil ity of p 6 0 s r c to phosphorylate casein at tyrosine residues (Sefton and Hunter, 1984). Treatment of RSV- trans-formed Chinese hamster ovary cells (CHO) with cholera toxin or 8-BromocAMP also stimulates the phosphorylation. of p 6 0 s r c at serine residues, concomitant with an apparent increase in kinase ac t i -vity (Roth, et a l . , 1983). Interestingly, p 6 0 s r c expressed and synthesized in E. col i i s not phosphorylated, yet i t possesses about 10% of the kinase activity assayed for p 6 0 s r c from eukaryotic cells (Gilmer and Erikson, 1981). These data suggest that phosphoryla-tion is not absolutely essential, but that i t may regulate the tyrosine kinase activity. An increase in cAMP levels in adipocytes has been correlated with a decrease in the binding of EGF and insulin by their respective receptors (Pessin, et a l . , 1983). Exposure of 3T3 cells to tumor promoters also results in a decrease in EGF binding by i ts receptor (Magun, et a l . , 1980). These treatments activate the cAMP dependent 2+ serine/threonine protein kinase or the Ca -diacylgylcerol - 33 -activated serine/threonine protein kinase C. Therefore, these effects may be mediated either directly or indirectly by the phosphorylation of these receptors at serine or threonine residues. Tumor promoters have also been shown to enhance the phosphorylation of both the insulin and IGF1 receptors (Jacobs, et a l . , 1983b), which suggests a possible role for Okinase in regulating these receptors as well. Recently, protein kinase C has been shown to phosphorylate the EGF receptor at a specific threonine residue (Hunter, et a l . , 1984), which subsequently reduces i t s EGF-stimulated tyrosine protein kinase activity (Cochet, et a l . , 1984). Taken together, these observations suggest that various func-tional properties of tyrosine kinases may be regulated by phosphoryla-tion on serine and/or threonine residues. 1.11 Fujinami Avian Sarcoma Virus Fujinami sarcoma virus (FSV) is an acutely oncogenic retro-virus, which was isolated from a naturally occurring chicken fibrosar-coma in 1909 by Fujinami and Inamoto (Fujinami and Inamoto, 1914). This virus primarily induces solid tumors in chickens and transforms fibroblasts in culture (Lee, et a l , 1980; Hanafusa, et a l . , 1980). Originally i t was reported that FSV did not cause any type of leukemia (Lee, et a l . , 1980; Hanafusa, et a l . , 1980); however, FSV has recently been shown to transform chicken erythroid cells both in vitro and in vivo (Kahn, et a l . , 1984). The transforming abil ity of FSV is due to the expression of an one gene, termed v-fps, that was acquired from - 34 -the normal chicken DNA by recombination with a proto-oncogene sequence called c-fps (Bishop and Varmus, 1982; Bishop, 1983; Varmus, 1984). These c-fps sequences were inserted into a FSV associated nontransform-ing retrovirus (FAV), where they have replaced the three essential replicative genes and a l l but the 5' end of the gag gene (Lee, et a l . , 1980; Hanafusa, et a l . , 1980). The insertion of v-fps into the FAV genome occurred in frame so that the v-fps gene became attached directly to the partially deleted gag gene. As a consequence of this recombination, FSV encodes a single gag-fps fusion protein and requires a helper virus such as FAV to replicate. 1.11.1 The FSV Genome The 4.5 kilobase (kb) FSV RNA genome contains a contiguous FSV-specific sequence flanked by 5' and 3' termini related to the RNA of the nondefective helper virus FAV (Lee, et a l . , 1980; Hanafusa, et a l . , 1980). The FSV genome has been recently cloned (Shibuya, et a l . , 1982b) and sequenced (Shibuya and Hanafusa, 1982). The structure of the FSV RNA genome inferred from the cloned DNA nucleotide sequence i s : 5' -Upj-leader-gag-f ps-C-Ug-R-31. The 5» 1.0 kb of the FSV genome contains a 21 base pair (bp) sequence that is repeated at both termini of the viral RNA (R), an 80 bp unique sequence (U5) and the leader sequence together with part of the FAV gag gene, which encodes the precursor virion core protein The internal 3.0 kb is FSV specific, since i t contains the v-fps gene which is essential for - 35 -the transforming activity of FSV. The 3' 0.5 kb of FSV RNA corresponds to the C or "common" region of the FAV RNA, and contains no known coding sequence, a 200 bp unique sequence ( U 3 ) and a 21 bp repeat (R). 1.11.2 Variant Strains of FSV The FSV genome encodes a single protein having a molecular weight (MW) of 140,000 daltons ( P i 4 0 g a g - f P s ) or 130,000 daltons (P130 g a g ~ f P s ) , depending upon the strain used. From the original FSV/FAV stock (Fujinami and Inamoto, 1914) a number of different strains have apparently arisen spontaneously, which encode transforming proteins of different size and temperature stability (Lee, et a l . , 1980; Hanafusa, et a l . , 1980; Hanafusa, et a l . , 1981; Lee, et a l . , 1981). Since characterization of the FSV transforming protein has involved the use of a number of different FSV strains, I w i l l refer to the transforming protein of FSV as either p i 4 0 g a g _ f P s or p 1 3 0gag-fps throughout this thesis. 1.11.3 The Relationship of FSV to Other Oncogenic Viruses FSV is one of a number of avian sarcoma viruses which have been grouped into four classes (Table 1.2) based on the relatedness of their cel l derived sequences (Wang, et a l . , 1982; Shibuya, et a l . , 1982a; Shibuya, et a l . , 1982b; Shibuya and Hanafusa, 1982; Huang, et - 36 -TABLE 1.2 Avian Sarcoma Viruses, Their Cell-derived Sequence Inserts and Gene Products Virus Class Cell-derived Transformation-Sequences Specific Protein Rous sarcoma virus (RSV) I Avian sarcoma virus B77 Fujinami sarcoma virus (FSV) II Avian sarcoma virus PRCII Avian sarcoma virus PRCIV Avian sarcoma virus UR1 Avian sarcoma virus 16L Avian sarcoma virus Y73 III Esh sarcoma virus (ESV) src fps yes p 6 0 s r c Pl^gag-fps piOs^g-fps pi70g a g- f PS Pl5 0gagrfps P142gag- fP s P90gag -yes pse^g-yes Avian sarcoma virus UR2 IV ros P68gag-yes - 37 -a l . , 1984; Neckameyer and Wang, 1984) and their encoded transformation specific proteins (Ghysdael, et a l , 1981; Patschinsky and Sefton, 1981; Neil , et a l . , 1982). The transforming protein of FSV is related structurally and functionally to at least six groups of acutely onco-genic retroviruses (discussed in section 1.6.2). A number of studies have suggested that c-fps and c-fes are the avian and feline versions, respectively, of the same genetic locus (Shibuya, et a l . , 1980; Barbacid, et a l . , 1981; Beemon, 1981; Shibuya, et a l . , 1982a; Groffen, et a l . , 1983). The viral forms of fps and fes share about 70% of their nucleotide sequences and deduced amino acid sequences (Shibuya and Hanafusa, 1982; Hampe, et a l . , 1982). Thus i t appears, that retro-viruses from two completely unrelated groups have acquired the equi-valent or related sequences from genomes of two distantly related species. 1.11.4 The FSV Encoded Transforming Protein The 4.5 kb FSV RNA is translated into a single 140,000 dalton protein which accounts for the entire coding capacity of FSV RNA in one reading frame (Lee, et a l . , 1980; Hanafusa, et a l . , 1980). Cell-free translation of FSV RNA has also revealed no FSV-specific products other than P 1 4 0 g a g ~ f p s , suggesting that i t i s the sole product of the FSV genome (Lee, et a l . , 1980; Pawson, et a l . , 1980). pi4o£ a g" f P s i s immunoprecipitated from transformed cells by antiserum raised against the virion gag proteins, but not by antiserum to the other - 38 -replicative gene products (pol and env) (Feldman, et a l . , 1980). These immunoprecipitation studies, along with tryptic peptide mapping studies of P l 4 0 g a g ~ f p s (Pawson, et a l . , 1980; Pawson, et a l . , 1981) and, more conclusively, the deduced amino acid sequence of the FSV trans-forming protein (Shibuya and Hanafusa, 1982) indicate that i t contains an N-terminal region synthesized from a defective gag gene, and a C-terminal region encoded by FSV-specific sequences (fps). Both genetic and biochemical data have suggested that P 1 4 0 g a g ~ f p s i s the transforming protein of FSV (Pawson, et a l . , 1980; Pawson, et a l . , 1981; Lee, et a l . , 1981; Hanafusa, et a l . , 1981; Lee, et a l . , 1982). p i 4 0 g a g ~ f p s isolated from transformed cells i s phosphorylated mainly at serine and tyrosine residues, and possesses a protein kinase activity specific for tyrosine residues (Pawson, et a l . , 1980; Feldman, et a l . , 1980; Pawson, et a l . , 1981). In an in vitro kinase reaction P 1 4 0 g a g - f p s can phosphorylate both i tsel f and exogenous substrates specifically at tyrosine residues. Cells transformed by FSV have enhanced phosphotyrosine levels compared with nontransformed cel ls, suggesting that P l 4 0 g a g - f p s functions as a tyrosine kinase in vivo. Studies with temperature sensitive (ts) mutants of FSV indicate that phosphorylation of P l 4 0 g a g _ f p s is necessary for init iation and maintenance of the transformed state of the cel l (Pawson, et a l . , 1980; Lee, et a l . , 1981; Hanafusa, et a l . , 1981; Lee, e t a l . , 1982). At the restrictive temperature the degree of - 39 -morphological transformation, total cel l phosphotyrosine levels, as well as phosphorylation of the transforming protein and i ts intrinsic kinase activity are a l l coordinately and reversibly decreased. 1.11.5 Cellular Location of the FSV Transforming Protein As discussed in section 1.8, the inappropriate phosphoryla-tion of key cellular proteins regulating cel l morphology, metabolism and growth could bring about the myriad of changes associated with transformation by FSV. One would anticipate that the intracellular location of pi-iO23-6"*1^8 would influence the structures, sub-strates, and regulators with which i t must interact to induce the process of ce l l transformation. A substantial fraction of P140g a g~-*-p s i s associated with the plasma membrane or cytoskeletal structures in FSV-infected cells; however, this association is salt sensitive (Feldman, et a l . , 1983; Moss, et al•, 1984). The P 1 4 0 g a g ~ f p s encoded by a ts mutant of FSV loses i t s membrane association at the nonpermissive temperature and becomes soluble (Moss, et a l . , 1984). This property of the ts P 1 4 0 g a g _ f p s suggests that i t s intracellular location may be cr i t ica l to i t s transforming capa-city. The identity and intracellular locations of the physiologically significant targets of p i 4 0 g a g ~ ^ p s are unknown. However, these studies suggest that the association of the FSV transforming protein with cellular components may be necessary for i t s interaction with - 40 -substrates whose phosphorylation i s required to induce and maintain cellular transformation. 1.11.6 The Normal Cellular fps Homologue Recently the normal cellular homologue of Pl40 g a g~-^P S , a 98,000 dalton protein (NCP98), was identified in uninfected chicken tissue (Mathey-Prevot, et a l . , 1982). NCP98 is homologous to the fps region of p i 4 0 g a g ~ f P s and has an associated tyrosine specific kinase activity. Curiously, NCP98 isolated from cells does not appear to be phosphorylated at tyrosine residues, unlike p i 4 0 g a g _ f P s . NCP98 is expressed in high levels in cells of the myeloid lineage (Shibuya, et a l . , 1982a; Mathey-Prevot, et a l . , 1982), suggesting a possible role in hematopoiesis. This is consistent with the idea that the normal cellular proteins encoded by the c-oncs are involved in cellular development and differentiation. Cell fractionation studies have revealed that NCP98 i s a predominantly soluble protein in E26-infected chicken myeloblasts, unlike i t s homologous transforming pi40g aS~ fP s > which i s mostly associated with cellular structural components in FSV transformed cells (Young and Martin, 1984). This difference in cellular location i s probably not due to the absence of gag sequences, since the transform-ing protein of the recombinant virus F36 (P92 fPs) lacks gag sequences (Foster and Hanafusa, 1983), and is also recovered in the - 41 -particulate fraction like P 1 4 0 g a g - f p s (Young and Martin, 1984). The results suggest that an alteration in the fps sequences is respon-sible for the different cellular localization between the FSV trans-forming protein and i ts normal cellular homologue, and that association with membrane or cytoskeletal structures i s required for trans-formation. 1.11.7 Structure of the FSV Gene Product For analysis, the transforming protein of FSV can be divided into three regions (Stone, et a l . , 1984): (1) the gag component (amino acid residues 1-309), which is encoded by sequences derived from FAV; (2) the C-terminal region (residues 888-1182), which is homologous to the C-terminal domain of p 6 0 s r c ; and (3) the middle or N-terminal fps-specific region (residues 310-388). A number of studies have been directed towards determining which regions of the FSV transforming protein are crucial for cellular transformation and tumorigenicity. The function of gag-protein determinant fused to the one gene product is not entirely understood. The gag protein does not appear to influence intracellular locations of the gag-one fusion protein, since p 6 0 s r c , which contains no gag protein sequences, and p g o ^ g - ^ 6 8 which does, are found at very similar intracellular locations. In addition, p 5 5 f o s , pnogag-W 0, p 48 m y b and p66^c are a l l nuclear proteins, regardless of the absence or presence of - 42 -gag sequences (RohrSchneider and Gentry, 1984). To examine the role the gag determinants play in cellular transformation, v iral DNA was constructed so that the src gene of RSV was replaced by greater than 90% of the fps sequence from FSV (F36 virus) (Foster and Hanafusa, 1983). Studies with the F36 recombinant virus have suggested that the gag determinants are not required for the transforming capacity, tumor-inducing abil ity or tyrosine kinase activity of the fps gene product of FSV. Although this data indicates that the gag sequences are not required for transformation, the fact that seven independent isolates of fps-containing acutely transforming retroviruses (FSV, PRCII, PRCIV, UR1, 16L, ST-FeSV, and GA-FeSV) express gag-linked fps-fusion trans-forming proteins suggests that the association is more than fortuitous (Bishop and Varmus, 1982 Neel, et a l . , 1982). In fact the transformed foci obtained with the gag-deleted F36 recombinant were morphologically more subtle than those obtained with FSV, suggesting that the gag region is not irrelevant to the activity of the FSV transforming protein. Also, deletion of gag sequences from the transforming gene of Abelson MuLV destroys the viruses abil ity to induce B-cell lymphomas in mice but the virus can s t i l l transform fibroblasts in cel l culture (Prywes, et a l . , 1983). In addition, in-phase insertion mutagenesis of the FSV genome has indicated that insertions in the gag region result in mutants with reduced focus forming activity, further supporting a role for gag in FSV transformation (Stone, et a l . , 1984). - 43 -The same linker-insertion mutagenesis study of the FSV genome has suggested that the N-terminal portion of the fps gene is important in fibroblast transformation (Stone, et a l . , 1984). However, mutations in this region of the protein have no effect on the catalytic activity of the mutant proteins, indicating that the sequences in the N-terminal portion of P 1 3 0 g a g - f p s are not involved in the phosphotransferase activity of the protein. Perhaps mutations in this area of the gene alter the subcellular location of the mutant protein and/or i ts inter-action with specific cellular substrates. The C-terminal region of P 1 3 0 g a g - ^ p s contains a region of shared homology with the other members of the src family of tyrosine kinases, as well as the other related oncogene products which lack detectable tyrosine kinase activity (section 1.6.2). Genetic and biochemical studies have provided evidence which suggest that this highly conserved region possesses the catalytic site for phosphotransferase activity. Several lines of evidence suggest that the structural and functional properties of the catalytic domain may be shared among different tyrosine kinases. First, a C-terminal proteolytic fragment of pi4oS a 6 - f P s possess kinase activity (Weinmaster, et a l . , 1983), as does the corresponding region of pgQsrc (Levinson, et a l . , 1981; Brugge and Darrow, 1984) and the EGF receptor (Basu, et a l . , 1984). Second, mutations which inactivate the protein kinase activity of these proteins map in this domain (Bryant and Parsons, 1984; Stone, et a l . , 1984). These mutagenesis - 44 -studies indicate and support the arguments that the catalytic domain is crucial in the transforming process. A number of other mutagenesis studies have suggested that the major tyrosine phosphorylation site in this domain is not required for activity, but that i t may modulate the function of these transforming proteins (Snyder, et a l . , 1983; Cross and Hanafusa, 1983; Weinmaster, et a l . , 1984; Snyder and Bishop, 1984). The catalytic domain also includes a putative ATP binding site which the oncogenic tyrosine kinases share with a number of different functional proteins (see section 1.6.2). Site-directed muta-genesis in the src gene at the codon for lysine-295, which reacts specifically with the ATP analogue FSBA (Kamps, et a l . , 1984), el imi-nates both the kinase and transforming activities ' of p 6 0 s r c , confirming the importance of the tyrosine kinase domain in transforma-tion (M. Kamps, personal communication; M. Snyder, personal communi-cation) . 1.12 Purpose and Experimental Approach The transforming protein of FSV is phosphorylated at several serine, tyrosine and threonine residues in transformed cells and possesses an intrinsic protein kinase activity specific for tyrosine residues. The phosphorylation of tyrosine residues within the FSV transforming protein probably results from autophosphorylation occurring either in trans (Mathey-Prevot, et a l . , 1984), c is , or both. - 45 -However, the identity of the kinases involved in the serine and threonine phosphorylations is unknown. Several lines of evidence suggest that various functional properties of tyrosine kinases may be regulated by phosphorylation (section 1.10). Understanding the regulation of the p i40 g a g ~ : f P s kinase activity is an important step in understanding the mechanism of transformation by FSV. In this study I have investigated the phosphorylation of FSV P 1 4 0 g a g ~ f p s in detail to establish the relationship between in vivo and in vitro phosphorylation of P 1 4 0 g a g - f p s . To map the multiple sites of phosphorylation on p i 4 0 g a g _ f p s , I have uti l ized different proteolytic enzymes to generate peptide fragments which were then localized relative to the intact protein, using double digestion experiments and tryptic peptide mapping of the various cleavage fragments (Chapter 3). Reversible phosphorylation of proteins is a major mechanism for the regulation of protein function (Krebs and Beavo, 1979). There-fore i t seemed reasonable to anticipate that the phosphorylation of tyrosine protein kinases may also regulate their activity. To determine whether tyrosine phosphorylation has any effect on the kinase and transforming activities of p l 3 0 gag- fps I have mutated the major site of tyrosine phosphorylaton within this protein, tyrosine-1073 (Chapter 4). - 46 -In order to understand more fully the biological role of PISO^B-^8 and i ts kinase activity, i t i s necessary to character-ize certain biochemical properties of this protein. One important property of any protein kinase is i t s substrate specificity. There-fore, I have investigated the specificity of the protein kinase intr in -sic to FSV P 1 3 0 g a g _ f p s by using oligonucleotide-directed muta-genesis to change the codon for tyrosine-1073 to those for the other commonly phosphorylated hydroxyamino acids serine and threonine (Chapter 5). A protein kinase that functions in phosphotransfer must possess a functional ATP-binding site. If the kinase activity int r in -sic to PlSC 6 3 , 6"-*- p s i s crucial for i t s transforming activity, one would predict that alterations in the P 1 3 0 g a g ~ f p s ATP-binding site would eliminate not only i t s kinase activity, but also i ts transforming activity. To test this hypothesis and to locate the ATP binding site within the catalytic domain of p l 3 0 gag- fps I have mutated the highly conserved lysine-950, which by homology with the src kinase is proposed to be involved either in the binding, positioning or catalysis of ATP (Kamps, et a l . , 1984) (Chapter 6). The data gained from these studies have allowed the ident i f i -cation of certain structural and functional domains within the trans-forming protein of FSV and have provided information concerning the molecular mechanisms of transformation by FSV. CHAPTER 2 2.0 Materials and Methods 2.1 Cells and Viruses The strain 15 of FSV described by Lee, et a l . (1981), is a clonal isolate from a stock obtained by P. Duesberg from H. Temin, which has been traced back to the original isolate of A. Fujinami. FSV pseudotyped by FAV, termed FSV(FAV), and FAV of this strain were obtained from P. Duesberg and provided by T. Pawson. A temperature resistant (tr) derivative, trFSV (Lee, et a l . , 1981) was also pseudotyped with FAV. Both of these FSV strains encode a 140,000 dalton protein ( P 1 4 0 g a g _ f p s ) . FSV clone 12 isolated from a different FSV stock by Hanafusa, et a l . , (1981) encodes a 130,000 dalton protein ( P 1 3 0 g a g - f p s ) . PRCII rescued with ring-neck pheasant virus was obtained from G.S. Martin. gs - chicken embryo fibroblasts (CEFs) were obtained from H & N Farms. Cells were infected with virus (5 x 107 to 1 x 10** focus-forming units per ml), passaged after 3 or 4 days, and used on day 4 or 5. Unless otherwise specified, a l l cells were maintained at 37°C, in 5% COv,. The rat-2 thymidine kinase minus (TK~) normal fibroblast l ine, obtained from W. Topp by J . Stone (Topp, 1981), was grown in 100 mm Falcon dishes containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicil l in and streptomycin at 37°C in a 5% <X>2 atmosphere. - 48 -2.2 Radio-labelling of Cells Cells were seeded at a density of 3 x 10^ cells in a 35 nm well ( 2 x 3 Linbro plate) and the following day were incubated with op ^P-orthophosphate (1.0-2.0 mCi/ml, carrier free; ICN Pharmaceuti-cals, Inc.) in 1.0 ml of phosphate-free DMEM containing 1-2% FBS. After the labelling period the plate was transferred to ice, the radio-active medium removed, and the cells were washed two times with ice-cold phosphate-buffered saline (138 mM NaCl, 2.68 mM KC1, 1.5 mM KH2PO4, 8 mM Na2HP04 [pH 7.2]) and then lysed in a total of 500 ul of lysis buffer (1% Nonidet P-40 [NP40], 0.5% sodium deoxycholate, 10 mM Tris-hydrochloride [pH 7.5], 100 mM NaCl, 1 mM EDTA, 2 mM ATP, and 1% [wt/vol] aprotinin [Sigma Chemical Co.]). The lysate was cen-trifuged at 4°C at 27,000 x g for 30 minutes, and the supernatant was recovered and incubated with the appropriate antiserum. Cells were labelled with [ S]methionine according to a similar protocol, except that the cells were incubated with [ S]methionine (100 uCi/ml, 1,000 Ci/mmol; Amersham Corp.) in 1.0 ml of methionine-free DMEM containing 1-2% FBS. After the labelling period the radioactive medium was removed, the cells were placed on ice, washed twice with Tris-saline and then lysed in 50 ul of lysis buffer as described above except the lysis buffer contained 1.0% sodium dodecyl sulfate (SDS) (cell lysis buffer). The cel l lysates were cleared and treated exactly as described above. - 49 -2.3 Immunoprecipitation Prespun cel l lysates were incubated on ice with the appro-priate antiserum (2 to 4 ul) for 30 minutes and with 15 volumes of a 10% suspension of Staphylococcus aureus strain Cowan I ((IgGsorb; the Enzyme Center) in cel l lysis buffer for a further 30 minutes. The immune complex was then pelleted in a microfuge and washed successively with 1 M NaCl - 10 mM Tris-hydrochloride (pH 8.0) - 0.1% NP40; with 100 mM NaCl -1 mM EDTA - 10 mM Tris-hydrochloride (pH 8.0) - 0.1% NP40 -0.1% SDS, and with 10 mM Tris-hydrochloride (pH 8.0) - 0.1% NP40. Immunoprecipitates of 0 , £ ,P-labelled cells were washed once more with 1.5 M NaCl - 1 mM EDTA -10 mM Tris-hydrochloride (pH 7.5) - 0.1% NP40. A l l of these steps were performed at 4°C. Immunoprecipitates were prepared for SDS polyacrylamide gel electrophoresis (SDS-PAGE) as described below. 2.4 Immune Complex Kinase Reaction Samples of 3 x 105 cells/ml from normal or transformed cells were lysed in kinase lysis buffer (1.0% NP40 - 20 mM Tris-hydro-chloride (pH 7.5) - 150 mM NaCl - 1 mM EDTA - 0.5% sodium deoxycholate) and immunoprecipitated as described above. The immune complex was washed twice in kinase lysis buffer and twice in 10 mM MnCl2 - 20 mM Tris-hydrochloride (pH 7.5) and incubated with 1-30 uCi of [^_32p]ATP (3,000 Ci/mmol; Amersham) in 35 ul of 10 mM MnCl2 -- 50 -20 mM Tris-hydrochloride (pH 7.5) at 20°C. After the incubation 500 ul of kinase lysis buffer was added to stop the reaction and the immunoprecipitate was washed three times with kinase lysis buffer. The pellet was resuspended in 50 ul of SDS sample buffer at 30°C for 10 minutes to disrupt the immune complex. The released proteins were recovered in the supernatant following centrifugation in a microfuge for 3 minutes. The samples were then either stored at -20°C or prepared immediately for gel electrophoresis. Phosphorylation of the exogenous substrate enolase was detected by the addition of 5 ug of enolase, that had been previously treated at 30°C for 5 minutes in 25 mM acetic acid (Cooper, et a l . , 1984a), to the immune complex kinase reaction which was then incubated at 30°C for 15 minutes. The reaction was stopped by the addition of 500 ul of kinase lysis buffer, the immune complexes were recovered by centrifugation in a microfuge for 3 minutes and subsequently prepared for electrophoresis. 2.5 SDS - Polyacrylamide Gel Electrophoresis Samples were heated at 100 °C for 3 minutes in SDS sample buffer (2% SDS, 5% 2-mercaptoethanol, 10 mM Tris-hydrochloride (pH 6.8), 10% [vol/vol] glycerol, .001% Bromophenol blue) and then electrophoresed using a SDS-polyacrylamide discontinuous buffer system described by Laemmli (1970). A 15 cm vertical slab gel apparatus (Richter Scientific; Vancouver, B.C.) contained a 4.5% polyacrylamide stacking gel and usually a 7.5% separating gel. These gels were - 51 -prepared from a stock solution of 29.2% wt/vol of acrylamide (BRL) and 0.8% wt/vol of N-N'-bis-methylene acrylamide (BRL). The final concen-trations in the separating gel were as follows: 0.375 M Tris-hydro-chloride (pH 8.8) and 0.1% SDS. The gels were polymerized chemically by the addition of 0.1% by volume of N, N, N',N1-tetamethylethylenedia-mine (TEMED) and 0.01% ammonium persulfate. The stacking gels contained 0.125 M Tris- hydrochloride (pH 6.8) and 0.1% SDS and were polymerized in the same way as for the separating gels. The electrode buffer (pH 8.3) contained 0.025 M Tris, 0.192 M glycine and 0.1% SDS. The solu-bilized proteins were subjected to electrophoresis at a constant power of 2 watts per gel through the stacker and 3 watts per gel through the separating gel. Electrophoresis was stopped when the bromophenol blue reached the bottom of the gel. After electrophoresis, gels of P-labelled proteins were either covered with Saran Wrap and exposed to film (Kodak XAR-5) while wet at 4°C or fixed and stained by soaking overnight in , 0.04% Coomassie Bri l l iant Blue, 7.5% acetic acid and 50% methanol, to locate molecular weight markers of known size. Gels were destained in several changes of 7.5% acetic acid and 23% methanol in dist i l led water. The destained gels were dried onto 3MM f i l te r paper (Whatman) using a Hoefer slab gel drier. The sensitivity of P detection was increased by using an intensifying screen (DuPont; Lightning Plus) with XAR-5 film at -80°C (Laskey and Mi l ls , 1977). Gels of [35S]methionine-labelled proteins were impregnated with En3Hance (New England Nuclear Corp.) before drying and exposed at -80°C (Bonner and Laskey, 1974), unless the proteins were to be - 52 -analysed further, in which case fluorography was omitted. The films were developed using a Kodak Mil - f i lm processor. Molecular weights of proteins were determined from a linear regression best f i t plot of the log of molecular weight versus the Rf of markers of known molecular weight electrophoresed on the same gel. To quantitate the radio-activity in specific proteins the appropriate bands were excised from dried gels and assayed by scinti l lation counting in the presence of PCS scintil lant (Amersham). 2.6 Partial Proteolytic Cleavage with p!5 and V8 Protease Cleavage of immunoprecipitated proteins was as described by Vogt, et a l . (1979). Briefly, the washed Immune complex was resus-pended in 35 ul of cleavage buffer (1.5 M NaCl - 1 mM EDTA - 10 mM Tris-hydrochloride (pH 7.5) - 0.1% NP40) and incubated for 30 minutes at 37°C with 10 ul of 10 mg/ml NP40-disrupted virions (Prague B. Rous sarcoma virus), and then a further 5 ul of NP40-disrupted virus was added. After a total of 60 minutes, 50 ul of 2 x SDS sample buffer was added to stop the reaction. The immune complexes were prepared for SDS polyacrylamide gel electrophoresis as described above. Limited proteolysis with V8 protease was performed in situ in SDS-polyacrylamide gels as described by Cleveland, et a l . (1977). oo Briefly proteins were labelled with in vivo or in vitro and purified after immunoprecipitation by SDS-polyacrylamide gel electro-phoresis. The pertinent bands were excised from wet gels, equilibrated - 53 -with buffer (125 mM Tris-hydrochloride (pH 6.8), 1 mM EDTA, 0.1% SDS), and applied to the sample well of a fresh gel with a 5-cm-long stacking gel and a 15% polyacrylamide separating gel cross-linked with 0.0867% bisacrylamide. The sample was overlaid, f i rs t with this buffer con-taining 20% glycerol and then with buffer containing 10% glycerol and V8 protease. Electrophoresis was performed at 2 watts until the dye front approached the separating gel, then the current and the cooling system were turned off for 30 minutes. Electrophoresis was then resumed at 3 watts. Gels were fixed, stained, dried and treated as described in section 2.5 2.7 Analysis of Tryptic Peptides 3 2 P or [3^S]methionine labelled proteins were separated by SDS-polyacrylamide gel electrophoresis and the pertinant bands were excised from the gel by using suitable alignment markers. Analysis of the tryptic peptides was essentially as described by Beemon and Hunter (1978) with a few modifications. Gel slices cut from wet gels were crushed directly or in the case of dried gels, the backing paper was removed from the dried gel bands which were then allowed to swell in a small volume of elution buffer (0.05 M NH4H003, 0.1% SDS). The swollen pieces were crushed and homogenized with the f lat end of a disposable 5 ml syringe plunger with more buffer being added when necessary. Including washings, the final volume of buffer used was 5.0 ml. The homogenate was made 5% in 2-mercaptoethanol, boiled - 54 -for 5 minutes, and then shaken overnite at 37°C in a shaking water bath to elute the labelled protein. The gel fragments were pelleted for 10 minutes at a setting of 7 on an IEC bench top cl inical centrifuge at room temperature. The supernatant was carefuly removed, and the gel fragments were washed for 2 hours at 37°C with 2 ml of elution buffer. The gel fragments were pelleted again, and the second supernatant was pooled with the f i r s t . A 25-ug amount of bovine gamma globulin was added as carrier to the pooled supernatants and mixed thoroughly. The protein was then precipitated by making the solution 20% in trichloro-acetic acid (TCA) and leaving at 4°C overnite. The precipitated pro-tein was recovered by centrif ugation for 30 minutes at 27,000 x g in a Beckman JA-21 rotor at 4°C. The tube was drained thoroughly by inver-sion, and the pellet was washed twice with ethanol chilled to -20°C and using the same centrifugation conditions. The dried pellet was dis -solved in 100 ul of formic acid and then 25 ul of methanol and 40 ul of performic acid (30% r^ Cv? and 98% formic acid [ 1 : 9 ] which had been incubated for 1 hour at room temperature) were added to the solubilized protein and incubated for 2 hours in an ice-slurry. The performic acid oxidization solution was diluted with 3 ml of water, frozen in a dry-ice/ethanol bath, and lyophilized in a Savant SpeedVac. The oxidized protein was digested with 5 ug of L-(l-tosylamido-2-phenyl) ethyl chloromethyl ketone(TPCK)-treated trypsin (Worthington) in 0.5 ml of 0.05 M NH4H003 for 6 hours at 37°C. The digest was diluted with 2.5 ml of water, frozen and lyophilized as described above. This procedure was repeated twice more until a l l the NH4HCO3 had been - 55 -removed. The digest was finally dissolved in 0.5 ml of pH 2.1 electro-phoresis buffer (see below) and lyophilized. The trypsin-digested protein sample was resuspended in 5 ul of electrophoresis buffer and carefully spotted in 1-2 ul aliquotes onto a 20-by-20 cm thin-layer cellulose plate (TLC) (0.1 mm; E. Merck Lab). After the sample had been applied, the plate was dampened with electrophoresis buffer, pH 2.1 (water - 88% formic acid - acetic acid, 90:2:8 by volume) and subjected to 1,000 volts for 60 minutes. During electrophoresis, cooling water was circulated beneath the plate to prevent over-heating and a glass plate was placed upon the thin-layer cellulose plate. After electrophoresis, the plate was air dried and further developed by ascending chromatography in N-butanol - acetic acid - water - pyridine (75:15:60:50, by volume) in the second dimension. O O was detected by exposing the plates to XAR-5 film at -80°C with the aid of an intensifying screen (Laskey and Mi l ls , 1977). Thin-layer cellulose plates containing [^S]methionine labelled q tryptic peptides were sprayed with En^ Hance (New England Nuclear) and exposed to film at -80°C (Bonner and Laskey, 1974). The pattern produced by the two-dimensional separation of tryptic peptides from the various proteins analysed were found to be reproducible. However, the mobilities of the individual tryptic pep-tides were not always identical, presumably due to variation inherent in the preparation of the sample and the mapping procedure. Nonethe-less, tryptic peptide analysis was useful in establishing relationships - 56 -among the different proteins examined. Comigration studies were performed to further substantiate the apparent similar or dissimilar mobilities of certain tryptic peptides from different proteins. This was done by separating the tryptic digests from the different proteins on the same TLC plate, and where applicable the results are reported in the text. In some cases individual tryptic peptides were further analyzed for their phosphoamino acid content (as described below in section 2.8) and the approximate location of the peptides within the intact protein was determined. Tryptic peptides characterized in this manner have been assigned a number, while tryptic peptides which were variable or only occurred in minor amounts were not characterized further and were not numbered. 2.8 Analysis of Phosphoamino Acids Normal or transformed cells growing in 35 mm wells were qp labelled for 12-16 hours with u^P-orthophosphate at a concen-tration of 1.0 mCi/ml in DMEM lacking phosphate but supplemented with 2% FBS (not dialyzed). In order to determine the total cellular phos-phoamino acids the method described by Cooper et a l . (1983c) was followed. Briefly, the monolayers were washed twice with cold buffered saline and the plates were drained thoroughly on ice. To the drained monolayers 0.3 ml of lysis buffer (1.0% NP40, 1.0% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.0, 1% Aprotinin, 2mM EDTA) was added and the cells were scraped with a plastic policeman and left at 4°C for 10 minutes to solubilize adherent structures com-- 57 -pletely. The cells were then scraped again and transferred to a 1.5 ml eppendorf tube. The samples were centrifuged at 20,000 x g at 2°C for 20 minutes in a JA-21 rotor (Beckman). The cleared supernatant was transferred to a 1.5 ml eppendorf tube containing 0.4 ml of NTE (0.1 M NaCl, 0.01 M Tris-hydrochloride, pH 7.5, 0.001 M EDTA) and 0.4 ml buffer-saturated phenol (redistil led), at room temperature. The sample was vortexed (ful l speed) for 30 seconds and centrifuged for 1 minute in a microfuge at room temperature and the aqueous layer was discarded. The phenol layer was reextracted once with 0.8 ml of NTE and the aqueous layer was removed carefully to recover the interface above the phenol phase which was subsequently transferred to a 30-ml glass tube (Corex) to which 13 ml of water and 2 ml of 100% wt/vol TCA was added. The sample was mixed well and allowed to stand at 0°C for 1 hour to precipitate the proteins which were recovered by centrifugation at 20,000 x g in a JA-21 rotor for 10 minutes at 2°C and the supernatant was decanted from the pellet of protein and detergent. The pellet was extracted with 5 ml of CHCL^ /MeOH (2:1) at room temperature with gentle shaking and the protein precipitate was collected by centrifuga-tion as before. The translucent protein pellet was air-dried, dissolved in 200 ul of 5.7 M HCI at 100°C for 2 minutes and transferred to a glass hydrolyzing tube with two, 100 ul washes of 5.7 M HCI. The proteins were hydrolyzed at 110°C for 90 minutes in a sealed glass tube. The hydrolyzed proteins were then lyophilized, resuspended in pH 1.9 buffer (88% formic acid-acetic acid - water; 50:156:1794) and 5 x 10^ Cerenkov counts were applied to a thin-layer cellulose plate along with 0.3-0.5 ug of unlabelled phosphoserine, phosphothreonine and - 58 -phosphotyrosine. After the sample had been applied and dried, the plate was dampened with pH 1.9 electrophoresis buffer and subjected to 1,000 volts for 180 minutes towards the anode. After electrophoresis the plate was dried and then re-wetted with pH 3.5 buffer (pyridine: acetic acid:water; 10:100:1890) and electrophoresed at pH 3.5 at 1,000 volts for 80 minutes towards the anode. After electrophoresis, the plate was dried and exposed to film with an intensifying screen at -80°C. Marker phosphoamino acids were identified by spraying the plates with a ninhydrin stain (0.1 gm ninhydrin, 70 ml ethanol, 21 ml acetic acid, 2.9 ml, 2,4,6-collidine) and gentle heating on a hot plate. To quantitate the radioactivity in specific spots on cellulose thin-layer plates, the cellulose was scraped off and counted in 10 ml of PCS (Amersham). The radioactivity in each spot was corrected for the background determined by scraping and counting a clear area on each plate. q o 0 / SP-labelled proteins eluted from gels, or individual tryptic peptides scraped from TLC plates and eluted from the cellulose with pH 2.1 buffer were acid hydrolyzed and their respective phos-phoamino acids were lyophilized, separated by electrophoresis, identi -fied and quantitated exactly as described above. 2.9 Transfection of DNA into Rat-2 Cells Replicative form (RF) DNAs of phage containing the various FSV genomes described in the text were digested with Sst l , electro-- 59 -phoresed on 0.5% agarose gels, and the 4.7 kbp FSV inserts were isolated by electroelution and concentrated by ethanol precipitation. 150 ng (5 pmoles) of each purified 4.7 kbp FSV DNA was incubated with 0.25 units of T4 DNA ligase for 1 hour at 22°C. Ligated FSV DNA and 10 ug of carrier rat-2 DNA was then coprecipitated with calcium phosphate and added to subconfluent rat-2 cells (Topp, 1981) in 100 mm dishes with DMEM containing 10% FBS as originally described by Graham and Van der Eb (1973) and modified by Wigler et a l . (1979). Briefly, DNA precipitates were formed by dropwise addition of 150 ng of ligated insert plus 10 ug rat-2 carrier DNA in 0.5 ml of 250 mM CaCl2 to an equal volume of a solution containing 50 mM Hepes, 1.5 mM NaHP04 and 280 mM NaCl (pH 7.05), while bubbling air into the mixture. In the case of cotransfection with the thymidine kinase (TK) gene of Herpes simplex type 1, (pTK-j^  (Enquist, et a l . , 1979), 50 ng of pTK-^  was added to the DNA solution along with the appropriate FSV insert and rat-2 carrier DNA and TK positive clones were identified by HAT selection (Graham, et a l . , 1980) (see below). Twenty-four hours post-transfection the medium was removed and replaced with fresh DMEM containing 5% calf serum and 0.5 uM dexa-methasone or in the case of HAT selection DMEM was supplemented with 1.0 x 10~5 M hypoxan thine, 4.0 x 10~7 M aminopterin, 1.6 x 10~^ M thymidine plus 10% fetal bovine serum (HAT). The medium was then changed every four to five days until termination of the experi-ment. Foci of transformed cells and HAT resistant colonies were isola-ted using cloning cylinders, expanded in mass culture and maintained in - 60 -DMEM containing 10% FBS or 5% calf serum with 0.5 uM dexamethasone. Soft agar colony formation was assayed using 5 x 104 cells seeded in DMEM containing 10% FBS and 0.3% wt/vol Bacto-Agar (Difco) in 60 mm dishes. Cells were photographed with a Wild photomicroscope. 2.10 Oligonucleotide-directed mutagenesis Digestion of the X - F S V ~ 2 vector DNA with Sstl (BRL), iso la -tion of the 4.7 kbp FSV insert, cloning into the Sst 1 restriction site of M13mpl0 RF DNA, transformation of E. col i JM101 (D lacpro, SupE, t h i l , F', pro AB+, l a c i + , lac Z DM15 traD36) and identification and propagation of recombinant phage were carried out as described elsewhere (Shibuya and Hanafusa, 1982; Zoller and Smith, 1982; Messing, 1983; Maniatis, et a l . , 1982; Zoller and Smith, 1983; Zoller and Smith, 1984). Recombinant phage were screened for FSV inserts in the correct orientation for mutagenesis by using the mutagenic oligo-nucleotide as the primer in dideoxynucleotide chain termination sequencing reactions (Sanger, et a l . , 1980). Mutagenic oligonucleotides were synthesized manually using solid-phase phosphite triester synthesis (Adams, et a l . , 1983) or on a Applied-Biosystems Model 380-A oligonucleotide synthesizing machine using controlled pore glass beads as the solid support and Applied-Biosystems reagents. Purification of oligonucleotides following synthesis was carried out using a 20% polyacrylamide-7 M urea sequenc-ing gel (40 x 20 x 0.05 cm) in TBE (50 mM Tris, 50 mM borate, 1 mM - 61 -EDTA, pH 8.3). The desired product was eluted from the gel by a crushsoak method using 0.5 M ammonium acetate, isolated, concentrated by ethanol precipitation and the sequenced confirmed by using the DNA sequencing technique of Maxam and Gilbert (1980). Phage DNA was mutagenized according to Zoller and Smith (1984), in that a second primer for DNA synthesis, the M13 universal sequencing primer (5'-CCCAGTCAOGACGTT-3') was included in the reaction in addition to and at the same molarity as the mutagenic oligonucleo-tide encoding the desired mutation (see Chapters 4, 5 and 6). This extension reaction was incubated for 8 hours at 15°C in the presence of the four deoxynucleoside triphosphates, E. col i DNA polymerase (large fragment) (BRL) and T4 DNA ligase (BRL) and was then used to transform competent E. col i JM101 cells directly without further purification. Phage from the resulting plaques were grown up in 1.5 ml cultures and their DNAs extracted and resuspended in 30 ul of 10 mM Tris-HCl pH 8.0, 1 mM EDTA (Zoller and Smith, 1984) Two ul was then spotted onto nitro-cellulose (Schleicher and Schuell) and screened by hybridization with the mutagenic oligonucleotide which had been 5'-end-labelled with q o ^P (Zoller and Smith, 1984). In order to sequence the region encompassing the mutation sites of the phage ssDNAs, I obtained from M. Smith an oligonucleotide primer for dideoxynucleotide chain termination sequencing (5'-CCTGAAGATGAAGAAGCT-3') which corresponds to nucleotides 3421-3438 of the FSV genome. Phage containing confirmed mutant or wild type FSV inserts were further plaque-purified and grown to high t i ter for production of phage RF DNAs, which were isolated by alkaline lysis - 62 -of infected bacteria and purified by centrifugation on CsCl gradients (Maniatis, et a l . , 1982). 2.11 Synthesis of p-Fluorosulfonylbenzoyl-5'-Adenosine FSBA was prepared according to Pal et a l . (1975). The compound had the same UV absorbance pattern described in the literature (Pal, et a l . , 1975). FSBA was stored dry at room temperature under dessication and was prepared for use by dissolving a measured amount in dimethylsulfoxide (DMSO) (Eastman). 2.12 Reaction of FSBA with P l 4 0 g a g ~ f P s Immunoprecipitates of FSV-L5 transformed CEFs were prepared as described in section 2.3 and washed once in kinase lysis buffer and once in buffer containing 10 mM Tris-hydrocholoride pH 7.0, 10% gly-cerol. Reactions with FSBA were carried out at 37°C in the last wash buffer. The reaction was initiated by addition of FSBA and was term-inated by addition of 2-mercaptoethanol to a f inal concentration of 50 mM. Aliquots were removed at the indicated intervals and were assayed for ATP:phosphotransferase activity as described in section 2.4. - 63 -CHAPTER 3 3.0 Mapping of Multiple Phosphorylation Sites Within the Structural  and Catalytic Domains of the Fujinami Avian Sarcoma Virus Trans- forming Protein. 3.1 Introduction The avian sarcoma viruses (ASVs) are a group of acutely oncogenic RNA tumor viruses which contain four distinct transforming genes (Table 1.2). The amino acid sequences of their gene products, predicted from DNA sequence data, show remarkable homology in their C-terminal 300 amino acids, whereas their aminc^termini are largely unrelated (Kitamura, et a l . , 1982; Shibuya and Hanafusa, 1982; Huang, et a l . , 1984; Neckameyer and Wang, 1985). Reflecting this structural relationship, the ASV transforming proteins are a l l associated with protein kinases specific for tyrosine residues and are themselves phos-phorylated at serine and tyrosine sites. There is much genetic and biochemical data to suggest that the ASV transforming proteins induce cellular transformation by modulating the control of ce l l growth, structure and gene expression through the pleiotropic effects of protein phosphorylation (see Chapter 1). The genomic FSV RNA encodes a 140,000 dalton protein (P140 g a g ~ f p s ) or a 130,000 dalton protein (PISO 8 9^"^ 8), depending upon the variant strain used (section 1.11.2), which is - 64 -synthesized from a defective gag gene and the fps gene (Hanafusa, et a l . , 1980; Lee, et a l . , 1980). The nondefective gag gene of a replication-competent virus encodes a precursor (Pr76 g a g) to the five virion core proteins (Vogt, et a l . , 1975). It has previously been shown that P140 g a g ~ f P s possesses an N-terminal 40K sequence corresponding to the gag proteins pl9, plO and part of p27, and a C-terminal 100K sequence synthesized from the fps gene (Pawson, et a l . , 1981). FSV P l 4 0 g a g _ f P s immunoprecipitated with antiserum directed against antigenic determinants in i t s gag or fps regions is i tsel f phosphorylated exclusively at tyrosine residues, after incubation in vitro with [ % - 3 2 P] ATP and Mn 2 + (Feldman, et a l . , 1980; Pawson, et a l . , 1980; Pawson, et a l . , 1981). In contrast, P 1 4 0 g a g ~ f p s isolated from transformed cells is phosphorylated mainly at serine, tyrosine and possibly threonine residues (Pawson, et a l . , 1981). However, the tryptic phosphopeptides of P 1 4 0 g a g - f P s phosphorylated in vitro are similar to those from in vivo-phosphorylated P 1 4 0 g a g _ f P s (Pawson, et a l . , 1981). The phosphorylation of the FSV transforming protein is com-plex and may well affect i ts activity and function. Since the kinase activity and transforming activity of P 1 4 0 g a g ~ f p s are presumably related, I have investigated the phosphorylation of FSV p i 4 0 g a g ~ f P s in detail . The aim of this study was to establish the relationship between in vitro and in vivo phosphorylation of p i40 g a g ~^P s , and to locate the phosphorylation sites within the different structural and functional regions of the protein. 3.2 Results 3.2.1 Tryptic Phosphopeptides of P140&2~£PB To identify the sites of phosphorylation in the FSV trans-forming protein, I have analysed the phosphopeptides produced by trypsin digestion of P l 4 0 g a g ~ f p s from the temperature sensitive (ts) L5 strain of FSV. pi40 g a S~ f P s was isolated by immunoprecipi-tation from FSV-transformed cells which was either metabolically 32 labelled with P-orthophosphate or phosphorylated in vitro in the immune complex with [ ^ - 3 2 P ] ATP as the phosphate donor. The sites phosphorylated in vivo were then compared with those labelled in vitro. FSV P l 4 0 g a g _ f p s i s phosphorylated exclusively at tyro-sine residues in the in vitro immune complex kinase reaction (Feldman, et a l . , 1980; Pawson, et a l . , 1980). Tryptic phosphopeptide analysis of p i 4 0 g a g ~ f p s phosphorylated in vitro revealed five labelled spots (Figure 3.1C) after electrophoresis at pH2.1 and chromatography in a butanol-acetic acid-pyridine buffer in a thin-layer cellulose sheet. Under some conditions of analysis spots 3a and 3b were not seen, and 3c was the major phosphorylated peptide. Reanalysis of purified peptide 3c under the experimental conditions used in Figure 3.1 generated peptides 3a and 3b, suggesting that these are derived from modification of 3c, and that spots 3a through 3c represent - 66 -Figure 3.1: Tryptic phosphopeptide analysis of FSV p i 4 0 g a g ~ f p s . FSV P l 4 0 g a g _ f P s was labelled with 3 2 P in vivo op by incubation of transformed CEFs with Pj[ and isolated by subsequent immunoprecipitation of the labelled protein with anti-pl9 serum or phosphorylated in vitro in an immune complex kinase reaction after immunoprecipitation from transformed CEFs with anti-pl9 serum. Gel-purified P l 4 0 g a g ~ f p s was then digested with trypsin and separated in two dimensions on thin-layer cellulose plates. An indicates the sample origin. Electrophoresis at pH2.1 was from left to right with an anode on the left , and chromatography was from bottom to top. Tryptic digests were as follows: A, tsFSV p i 4 0 g a g ~ f P s , phosphorylated in vivo (18 hour 3 2 P i labelling); B, tsFSV p i 4 0 g a g ~ f P s phos-qp phorylated in vivo (4 hour Pj_ labelling); C, tsFSV p i 4 0 g a g ~ f P s phosphorylated in vitro. Phos-phoamino acid analysis of tryptic peptide from in vivo-labelled p i 4 0 g a g " f P s showed that spots 1, 3a through 3c, 4, and 6 contain predominantly phospho-tryosine, whereas spot 5 contains predominantly phospho-serine. - 68 -different forms of the same peptide. In addition cleavage of peptides 3b and 3c with Staphylococcus aureus V8 protease, as described by Patschinsky et a l . (1982), produced peptides which comigrate in the 2-dimensional system described above suggesting that they are derived from the same tryptic peptide (data not shown). In contrast, the re la -tively minor tryptic phosphopeptides 1 and 2 apparently represent d is -tinct sites of tyrosine phosphorylation (see below), suggesting that P140gag-fps i s phosphorylated in vitro at three different tyrosine residues. To compare the P i 4 0 g a g - : l " p s sites phosphorylated in vitro by i ts intrinsic kinase activity with the residues actually phosphory-lated in the transformed c e l l , p i 4 0 g a g - f p s was isolated from ^P-labelled ts FSV L5-transformed chicken embryo fibroblasts (CEF) and subjected to tryptic phosphopeptide analysis (Figure 3.1A and B). Secondary analysis of p i 4 0 g a g - f p s in vivo labelled tryptic peptides for phosphoamino acid content indicated that there were five major phosphotyrosine-containing spots, of which four (peptides 1 and 3a through 3c) were shown to comigrate with those from in vitro-phos-phorylated P 1 4 0 g a g - f p s . A major new tryptic phosphopeptide (spot 4) which contains phosphotyrosine as i ts sole phosphoamino acid is pre-sent in the digest of in vivo-labelled P 1 4 0 g a g - f p s , and peptide 2 of in vitro-phosphorylated p i 4 0 g a g - f p s is missing. Mixing experi-ments on tryptic digests of p i 4 0 g a S ~ f p s phosphorylated in vivo and in vitro show that peptides 2 and 4 migrate differently (data not shown). At least two minor tryptic phosphopeptides of in vivo-labelled - 69 -P 1 4 0 g a g - f P s comigrate with those found in FAV Pr76 g a g iso la -ted from 3 2 P-labelled, FSV (FAV)-infected cel ls , indicating that they represent normal sites of gag phosphorylation. In addition, P140gag-fps possesses a strongly labelled tryptic phosphopeptide containing phosphoserine (spot 5) which is not found in FAV Pr76 g a g and thus represents specific phosphorylation of a serine residue on P 1 4 0 g a g " f p s . These results indicate that ts FSV L5 P140gag-fps i s phosphorylated in transformed cells on three tyro-sine residues (contained within tryptic peptides 1, 3a through 3c and 4), of which one (peptide 4) is not phosphorylated in vitro, and on several serine residues of which at least one (peptide 5) is specific to P 1 4 0 g a g - f p s . Two other FSV-specific tryptic phosphopeptides from in vivo-phosphorylated pi40 g a S~ f P s (peptides 6 and 7) have not been analysed in any detail , although peptide 6 is known to contain phosphotyrosine. There is l i t t l e radioactivity remaining at the origin in these two-dimensional analyses and very l i t t l e free phosphate, ind i -cating that the majority of tryptic phosphopeptides have been separ-ated. Peptide 1 must have an overall negative charge, as i t migrates to the positive electrode at pH2.1, and i t therefore is either very small, multiply phosphorylated or contains cysteic acid in addition to a phosphate group since the side chains of glutamic acid and aspartic acid are not ionized at this pH. A l l of the other peptides migrate toward the negative electrode in the eletrophoretic dimension. For in vivo-phosphorylated pi4oS a g - f P s the relative labelling of the tyrosine residues within tryptic phosphopeptides 1, 4, - 70 -and 3a through 3c is approximately 0.2:0.6:1 (where the figure for 3a through 3c is the sum of these spots), although i t i s diff icult to know whether this reflects the actual steady-state levels in vivo. On in vitro-phosphorylated P 1 4 0 g a g ~ f p s , spots 3a through 3c represent a single major site of tyrosine phosphorylation, with tryptic phospho-peptides 1 and 2 comprising relatively minor phosphorylation sites. 3.2.2 Localization of Phosphorylation Sites on P l 4 0 g a g " f P s Proteolytic enzymes were used to cleave FSV p i 4 0 g a g - f p s into two or more fragments, and these fragments have been mapped onto the intact protein. By constructing such a proteolytic cleavage map of FSV P l 4 0 g a g - f p s I have been able to localize the various phosphor-ylation sites to different regions of the protein (see Figure 3.8). The cleavage of p i 4 0 g a g - f p s by the avian retrovirus virion pro-tease pl5 has been previously described (Von der Helm, 1977; Vogt, et a l . , 1979; Pawson, et a l . , 1981). pl5 cuts P 1 4 0 g a g ~ f p s within i t s N-terminal gag region, yielding a 33K, N-terminal, gag-encoded fragment [N-33K(pl5)] and a C-terminal, 120K fragment [C-120K(pl5)] that contains a small region of gag p27 sequence and the entire C-terminal fps-encoded portion (Pawson, et a l . , 1981). To localize phosphorylation sites to these two fragments, p i 4 0 g a g - ^ p s was 32 labelled in vivo with P-orthophosphate or phosphorylated in vitro in the immune complex reaction and then cleaved with pl5 by the - 71 -addition of disrupted Rous sarcoma virus virions to the immunopre-cipitated protein. Figure 3.2 shows that both the N-33K(pl5) and the O120K(pl5) fragments of in vivo- and in vitro-phosphorylated pi 4 0gag-fps a r e labelled with 3 2 P . Phosphoamino acid analysis of the N-terminal 33K fragment of in vitro-phosphorylated P 1 4 0 g a g _ f p s revealed only phosphotyrosine whereas the correspond-ing 33K fragment from P 1 4 0 g a g - f p s phosphorylation in transformed cells contained phosphoserine and phosphotyrosine in equivalent amounts and a trace amount of phosphothreonine (data not shown). Tryptic phos-phopeptide analysis of the N-terminal 33K gag-fragment of P 1 4 0 g a g - f p s phosphorylated in vivo yielded the acidic phospho-tyrosine-containing tryptic peptide 1 in addition to phosphopeptides which comigrate with those of FAV Pr76 g a g (Figure 3.3A). The N-33K(pl5) fragment of in vitro-phosphorylated P l 4 0 g a g ~ f p s i s labelled only at tryptic phosphopeptide 1 (Figure 3.3B). The C-terminal 120K pl5 cleavage fragment of in vivo-phosphorylated P 1 4 0 g a g ~ f p s gives phosphotyrosine-containing tryptic peptides 3a through 3c and 4 and phosphoserine-containing peptide 5 (Figure 3.3D), whereas, the C-120K(pl5) fragment of P 1 4 0 g a g _ f p s labelled in the immune complex reaction contains only peptides 3a through 3c (Figure 3.3E). These results indicate that P 1 4 0 g a g ~ f p s i s phosphorylated in transformed cells at a tyrosine site (tryptic phosphopeptide 1) within the gag region in addition to sites of phosphorylation shared with FAV Pr76 g a g . However, the major sites of tyrosine (peptides - 72 -Figure 3.2: Cleavage of FSV p i 4 0 g a g ~ f p s with pl5. FSV P l40gag-fps rag labelled in vivo with 3 2 P i and isolated by immunoprecipitation with anti-pl9 serum or phosphorylated in an immune complex kinase reaction with [^- 3 2P]ATP. Both samples were washed exten-sively, incubated at 37°C for 30 minutes in the presence or absence of 10 ug of NP40-disrupted RSV(Pr-B), and then prepared for electrophoresis on a 7.5% SDS-poly-acrylamide gel. Lanes: A and B, tsFSV P l 4 0 g a g ~ f p s phosphorylated in vivo (18 hour labelling); A, uncleaved; B, cleaved; C and D, tsFSV P l 4 0 g a g - f p s phosphorylated in vitro; C, uncleaved; D, cleaved. - 73 -4i PI D P140-H& m | — -«-120K : -*-33K - 74 -Figure 3.3: Tryptic peptide analysis of pl5 cleavage fragments of FSV p i 4 0 g a g _ f P s . 3 2 P - or 3 5 S-labelled polypeptides were gel purified, digested with trypsin, and analysed by two-dimensional separation on thin-layer cellulose plates. Protein fragments analysed were as follows: A. N-33K(pl5) obtained by pl5 cleavage of tsFSV P 1 4 0 g a g - f P s phosphorylated in vivo; B, N-33K (pl5) obtained by pl5 cleavage of tsFSV p i 4 0 g a g ~ f p s phosphorylated in vitro; C, N-33K(pl5) obtained by pl5 cleavage of tsFSV p i 4 0 g a g ~ f P s labelled with qc [OJS]methionine in vivo; D, C-120K(pl5) obtained by pl5 cleavage of tsFSV p i 4 0 g a g ~ f P s phosphorylated in vivo; E, O120K(pl5) obtained by pl5 cleavage of tsFSV P 1 4 0 g a g " f P s phosphorylated in vitro; F, FSV pi 4 0gag-fps isolated from tsFSV-transformed CEFs labelled with [3 5S] methionine; G, P r76 g a g 35 isolated from the same [ S]methionine-labelled cel ls . The numbering of methionine-containing peptides is according to Pawson et a l . (1981) and is differen-tiated by a superscript "S". Tryptic peptides I s through 5 s are fps specific. - 75 -• - 76 -3a through 3c and 4) and serine (peptide 5) phosphorylation are con-tained within the fps region. To verify the identity of the pl5 cleavage fragments used in these experiments, [35S]methionine-labelled p l 4 0 g a g - f P s isolated from FSV-infected cells was cleaved with pl5 and coelectrophoresed with the P-labelled fragments. The [35S]methionine-labelled 33K pl5 fragment, intact FSV P 1 4 0 g a g _ f p s , and FAV VrlQ^ were then subjected to tryptic peptide analysis (Figure 3.3). This confirmed that the 33K fragment contains only gag-encoded sequences. To localize phosphorylation sites in C-120K(pl5) more accur-ately within the fps-encoded region of P l 4 0 g a g - f p s partial proteo-lyt ic cleavage fragments were generated with Staphylococcus aureus V8 protease (Houmard and Drapeau, 1972) using the Cleveland gel technique (Cleveland, et a l . , 1977). Digestion of 3 2 P-labelled P140 g a g ~^ p s with low concentrations of V8 protease yields two major cleavage products with apparent molecular weights of 78K and 61K [78K(V8) and 61K(V8)] (Figure 3.4). Limited V8 protease digestion of [35S]methionine-labelled p i 4 0 g a g _ f P s also produces these two major cleavage fragments (Figure 3.5). Tryptic peptide analysis of the OK V8 protease digestion products of [ Sjmethionine-labelled P 1 4 0 gag-fps ( F i g u r e 3. 6) showed that the 61K(V8) fragment contains a l l of the tryptic peptides previously identified as fps specific (Pawson, e t a l . , 1981) (Figure 3.3). There is no apparent overlap - 77 -Figure 3.4: Cleavage of FSV pi40 g a g~ f P s with V8 protease. Lanes: A through C, tsFSV p i 4 0 g a g - f p s labelled 32 with P i during an immune complex kinase reaction, purified by SDS-polyacrylamide gel electro-phoresis, and then subjected to in situ digestion with V8 protease in a new gel with a 15% polyacrylamide separating gel; A, 500 ng of V8 protease; B, 100 ng of V8 protease; C, 50 ng of V8 protease. - 78 -A B C MW - 79 -Figure 3.5: Cleavage of r°S]methionine or J Z P-label led FSV p i 4 0 g a g - f P s with V8 protease. 3 2 P - or 35 S-labelled proteins were isolated from prepara-tive gels, and subjected to in situ digestion with V8 protease (50 ng per well) followed by electrophoresis through a 15% SDS-polyacrylamide separating gel. Lane 1, tsFSV PMO^S^P*3 labelled with 3 2 P in vitro; Lane 2, [35S]methionine-labelled tsFSV p1 4 0gag-fps obtained by cell -free translation of FSV(FAV) poly(A)-selected heat-denatured 70S virion RNA in a messenger-dependent rabbit reticulocyte lysate. - 80 -1 2 - 81 -between these two V8 protease cleavage fragments, arguing that they represent a unique N-terminal 78K fragment and a unique C-terminal 61K fragment presumably separated at a single V8 protease cleavage site in the middle of the P M O ^ ^ P 8 1 region (Figure 3.6 A and D). To test this deduction, I isolated the O120K(pl5) fragment of in vitro-phosphorylated pi4oS ag~ fP s and digested i t with V8 protease (Figure 3.7); this generated the same 61K(V8) fragment contained within the intact P l 4 0 g a g ~ f P s , but no 78K(V8) fragment. This would be expected i f 78K(V8) corresponded to the N-terminal half of p 1 4 0gag-fps a n d 6 1 K ( V 8 ) t o i t s c-terminal half since C-120K(pl5) has lost most of the gag sequence but retains a l l of the C-terminal fps sequence. The N-terminal 33K(pl5) fragment is resistant to limited V8 protease digestion. Tryptic phosphopeptide analysis of the N-terminal 78K and C-terminal 61K V8 protease fragment [N-78K(V8) and C-61K(V8)] of P 1 4 0 gag-fps f r o m 3 2 P-labelled ts FSV L5-transformed cells showed that the C-61K(V8) fragment contains peptides 3a through 3c, 4 and 5, whereas, the N-78K(V8) fragment contains peptide 1 (Figure 3.6 B and C). This corroborates the localization of phosphotyrosine- con-taining tryptic peptide 1 to the N-terminal gag region and indicates that the two major phosphotyrosine sites (peptides 3a through 3c and 4) and the major phosphoserine site (peptide 5) are clustered in the C-terminal portion of p i 4 0 g a g - f P s . A similar analysis of Pl40 g a g ~^P s phosphorylated in vitro in the immune complex kinase - 82 -Figure 3.6: Tryptic peptide analysis of V8 protease cleavage frag-ments of FSV p i 4 0 g a g ~ f P s . 3 2 P - or 3 5 S -labelled polypeptides were gel purified, digested with trypsin, and analysed by two-dimensional separation on thin-layer cellulose plates. Protein fragments analysed were as follows: A, N-78K(V8) produced by V8 cleavage of [35S]methionine-labelled tsFSV pi4o8ag-fps obtained by cell -free translation of FSV(FAV) poly-adenylic acid-selected, heat-denatured 70S virion RNA in a messenger-dependent rabbit reticulocyte lysate; B, N-78K(V8) obtained by V8 protease cleavage of tsFSV P 1 4 0 g a g _ f P s phosphorylated in vivo (18 hour label-ling); C, N-78K(V8) obtained by V8 protease cleavage of tsFSV p i 4 0 g a g - f P s phosphorylated in vitro; D, C-61K(V8) produced by V8 protease cleavage of [35S]methionine-labelled tsFSV P i 4 0 g a g _ f P s obtained by cell -free translation of FSV(FAV) poly-adenylic acid-selected, heat-denatured 70S virion RNA in a messenger-dependent rabbit reticulocyte lysate; E, C-61K(V8) obtained by V8 protease cleavage of tsFSV P140 g a g~^P s phosphorylated in vivo (18-hour label-ling); F, C-61K(V8) obtained by V8 protease cleavage of tsFSV p i 4 0 g a g _ f P s phosphorylated in vitro. The numbering of methionine-containing peptides is according to Pawson, et a l . (1981) and is differentiated by a superscript "S". Tryptic pepties I s and 5 s are fps specific. - 84 -Figure 3.7: Mapping V8 protease cleavage fragments of FSV P140gag-fps# T h e t s F S V p i 4 0 e a g - f P s was 32 labelled with P^ during an immune complex kinase reaction and cleaved with NP40-disrupted virions. Uncleaved pi40gag~ fP s and the two pl5 cleavage fragments 120K(pl5) and 33K(pl5) were recovered from the same gel and then subjected to in situ V8 protease digestion with 50 ng of V8 protease per sample followed by electrophoretic separation on a 15% SDS-polyacryla-mide gel. Lanes: A , tsFSV P M O S 3 - 6 " * 1 * 3 ; B , 120K(pl5) fragment; C, 33K(pl5) fragment. A B C - 86 -reaction (Figure 3.6 C and D) demonstrates that N-78K(V8) contains tryptic phosphopeptides 1 and 2, whereas C-61K(V8) contains only tryptic phosphopeptides 3a through 3c. This concurs with the observa-tion that the N-78K(V8) from in vitro-phosphorylated P l ^ ^ S - * ? 8 i s more heavily labelled with 3 2 P relative to C-61K(V8) than the corresponding fragment from in vivo-phosphorylated pi4QB&&~fPs (data not shown). It i s possible that the in vitro-phosphorylated peptide 2 is near the site of pl5 cleavage, since i t is not easily recovered in the pl5 cleavage fragments. The results of these mapping experiments are shown diagram-atically in Figure 3.8. 3.2.3 Phosphorylation of the Transforming Proteins of Different fps  Viruses FSV L5 induces a temperature-sensitive, transformed phenotype and encodes a PMO6 3-8""-^8 with a thermolabile protein kinase ac t i -vity (Pawson, et a l . , 1980). To determine whether this temperature sensitivity reflects any change in the amino acids sequence immediately surrounding p i ^ S 3 , 6 " - ^ 8 phosphorylation sites, I analysed the tryptic phosphopeptides of i t s temperature-resistance derivative, tr FSV (Lee, et a l . , 1981). Figure 3.9 shows that t r FSV and ts FSV P M O ^ S - ^ 8 , isolated from FSV-transformed CEFs metabolically labelled with 32P-orthophosphate, have tryptic phosphopeptides - 87 -Figure 3.8: Cleavage sites for pl5 and V8 protease on FSV P 1 4 0 g a g - f p s yielding the fragments described in the text. The numbers indicate the putative location of tryptic phosphopeptides within FSV p i 4 0 g a g - f P s as discussed in the text. 78 k- V8- 61k P15 33k 120k-4 00 00 NH, 1 2? 4 , 5 , 3a-3c COOH " gag— p19, p10, *p27 fps - 89 -Figure 3.9: Tryptic phosphopeptide analysis of trFSV P 1 4 0 gag-fps > FSV pi3o g a g - f P 8 and PRCII P105 gag- fP 8. FSV PWOS^-fP 8, FSV p 1 3 0 g a g - f p S ) o r P R C I I p l 0 5 gag- fps was labelled •30 with °^ P in vivo by incubation of transformed CEFs with °~Pi and isolated by subsequent immunopreci-pitation with anti-pl9 serum, or labelled in vitro by phosphorylation in an immune complex kinase reaction with [)(-32P]ATP after immunoprecipitation with anti-pl9 serum from transformed CEFs. Gel-purified P 1 4 0 gag- fps ) p l 3 0 gag- fp S ) o r p l 0 5 gag- fps was then digested with trypsin and separated in two dimensions on thin- layer cellulose plates. Tryptic digests were as follows: A, trFSV p i 4 0 g a g - f P 8 phosphorylated in vivo; B, FSV P 1 3 0 g a g ~ f p 8 phos-phorylated in vivo; C, trFSV PMO 2 3 - 6 "^ 8 phosphory-lated in vitro; D, PRCII P105 g a g ~ f P 8 phosphorylated in vitro; E, PRCII P 1 0 5 g a g " f p 8 phosphorylated in vivo. The arrows indicate normal migration of spot 4 as determined by a mixing experiment. The identity of spots is based on comigration in mixing experiments with tsFSVLo P 1 4 0 g a g ~ f p s . The additional spots in panels B and E have not been analysed further. - 91 -identical to those of their p i 4 0 g a g - f p s proteins phosphorylated in the immune complex (peptide 1 and peptides 3a through 3c). Another variant of FSV with a different passage history (Hanafusa, et a l , 1980; Hanafusa, et a l . , 1981) was also analysed. The P ISO 8 3 , 6 - ^ 8 from this variant is phosphorylated in vivo with 3 2 P at phosphotyrosine containing tryptic peptides which have similar mobilities with those of ts FSV L5 and tr FSV (Figure 3.9B). However, new phosphoserine-containing peptides are seen, suggesting that there has been either sequence divergence in the tryptic peptide encompassing the major ser-ine phosphorylation site, or that the sites are completely different. PRC! I is an independent isolate of a transforming virus con-taining fps sequences whose encoded protein lacks a sequence of 340 amino acids that is found in the N-terminal half of the fps domain of FSV P 1 3 0 g a g - f p s (Huang, et a l . , 1984). PRCII P 1 0 5 g a g ~ f p s phosphorylated in the immune complex kinase reaction has a tryptic phosphopeptide map similar to that of in vitro-phosphorylated tsFSV L5 and trFSV P 1 4 0 g a g - f p s , suggesting that i t has retained the sequences encoding the conserved tyrosine phosphorylation sites 1 and 3a through 3c (Figure 3.9D). However, PRCII P 1 0 5 g a g - f p s phosphor-ylated in vivo is labelled at tryptic phosphopeptides 1 and 3a through 3c, but not or very weakly at the phosphotyrosine site represented by tryptic phosphopeptide 4 found in the FSV transforming proteins examined (Figure 3.9E). It i s therefore possible that this site is contained within a region of FSV p i 4 0 g a g - f p s that is deleted in PRCII P 1 0 5 g a g - f p s . - 92 -3.3 Discussion Phosphoamino acid analysis of FSV p i 4 0 g a g _ f p s indicates that i t is phosphorylated in transformed cells at multiple tyrosine residues. Comparative tryptic phosphopeptide analysis suggests that these residues are contained within a region which is highly conserved between the transforming proteins of different FSV variants. The N-terminal gag region of p i 4 0 g a g - f p s contains minor phosphorylated sites shared with Pr76 g a g (Pawson, et a l . , 1981 and this study), but surprisingly i t is also phosphorylated at a tyrosine residue con-tained within an acidic tryptic phosphopeptide. The importance of this gag phosphotyrosine site to the functional activity of p i 4 0 g a g - f p s i s unknown. Clearly, the tyrosine phosphorylation within the gag is specfic, but whether this represents fortuitous phosphorylation owing to the proximity of this sequence to the kinase active site or an important functional modification is yet to be determined (see section 1.11.7). The major fps-specific phosphorylation sites of FSV P 1 4 0 g a g - : f p s , including two phosphotyrosine residues and a phosphoserine residue, are clustered in the C-terminal region of fps, as represented by the 61K C-terminal V8 protease fragment. Limited trypsin cleavage experiments indicate that this region of the protein contains the kinase active domain (Levinson, et a l . , 1981; Weinmaster, et a l . , 1983; Brugge and Darrow, 1984). The C-terminal localization of the major fps phosphorylation sites and the detection of trypsin fragments with kinase activity support the suggestion that - 93 -the C-terminal sequences of the ASV transforming proteins are highly conserved because they encode the kinase domain. The conservation of sequence surrounding the p i 4 0 g a g ~ f P s phosphotyrosine residues argues that these sites are important for activity of the protein and in the following two chapters I w i l l present data to support a relationship between the phosphorylation of p i 4 0 g a S - f P s i tself and i ts kinase activity. Several groups have compared the phosphorylation sites from a number of different v i ral transforming proteins by using micro-sequencing (Neil, et a l . , 1981; Neil, et a l . , 1982; Patschinsky, et a l . , 1982) and deduced amino acid sequences (section 1.6.1). There is substantial homology between the characteristically acidic amino acid sequences N-terminal to the tyrosine phosphorylation sites (see figure 1.1). For PRCII P 1 0 5 g a g ~ f p s and FSV p i 4 0 g a g - f P s phosphorylated in vitro, the major tyrosine phosphorylation site has a glutamic acid four residues N-terminal to the phosphotyrosine and a basic amino acid seven residues N-terminal (Patschinsky, et a l . , 1982). Examination of the amino acid sequence of FSV PISO^ 9^ -^ 8 deduced from the DNA squence of cloned FSV shows a tyrosine at residue 1073 which f u l f i l l s these characteristics (Shibuya and Hanafusa, 1982). The N-terminal amino acid of the tryptic peptide containing this tyrosine residue is glutamine, as in i t ia l l y suggested to account for poor yields in microsequencing (Neil, et a l . , 1982). Cyclization of the glutamine residue following hydrolysis of i ts side chain amide group during the - 94 -tryptic mapping procedure may yield a tryptic peptide having pyro-glutamic acid at i ts N-terminal end (3b) which is a less positively charged species at pH2.1 compared to the unmodified peptide containing glutamine at i ts N-terminus (3c). In addition, the pyro-glutamate ring may open to produce glutamic acid at the N-terminal end, which would be more negatively charged (3a). These observations and those discussed previously suggest that the tryptic peptides 3a through 3c, which represent the major site of tyrosine phosphorylation in vitro, correspond to the tryptic peptide containing tyrosine-1073. P140 g a g~*P s is also phosphorylated in vivo at a C-termi-nal tyrosine residue contained within peptide 4. It is intriguing that this residue is not phosphorylated in the immune complex kinase reac-tion, perhaps this site of tyrosine phosphorylation is not the product of pi40g ag~^P s autophosphorylating activity but rather is phos-phorylated by a cellular tyrosine kinase. Alternatively, the lack of phosphorylation within peptide 4 could reflect the a r t i f i c ia l nature of the in vitro immune complex kinase assay which would also account for the phosphorylation of peptide 2 which is only seen with P140gag~"^Ps phosphorylated in vitro. This lack of phosphorylation within peptide 4 does not affect the apparent kinase activity of P140 g a g~^P s as measured in vitro. It appears doubtful whether peptide 4 is present in PRCII P105 g a g ~ f P s . However, i f i t is absent from this transforming protein i t clearly does not affect i ts in vitro tyrosine protein kinase activity, but whether i t has anything - 95 -to do with the decreased oncogenicity reported for PRCII (Breitman, et a l . , 1981) remains to be seen. These data raise the possibility that more than one kinase is involved in the phosphorylation of P l 4 0 g a g _ f p s tyrosine residues in FSV transformed cel ls. Experiments described in the following chapters were designed to investigate the relationship between the phosphorylation of the FSV transforming protein and i ts enzymatic and biological activities. CHAPTER 4 4.0 Oligonucleotide-directed Mutagenesis of Fujinami Sarcoma Virus:  Evidence that Tyrosine Phosphorylation of p1 3 0gag-fps Modu-lates i ts Enzymatic and Biological Activit ies. 4.1 Introduction A variety of genetic and biochemical data indicate that the integrity of the P 1 3 0 g a g - f P s kinase function is essential for FSV to induce neoplastic transformation of infected cells (Pawson, et a l . , 1980; Hanafusa, et a l . , 1981; Lee, et a l . , 1981; Stone, et a l . , 1984). The aberrant phosphorylation at tyrosine of cellular proteins normally involved in regulating cel l growth and metabolism might explain many phenotypic changes associated with expression of the viral transforming protein. A number of cellular proteins do become newly or increasingly phosphorylated at tyrosine upon transformation by FSV, of which three have been positively identified as glycolytic enzymes (enolase, phos-phoglycerate mutase and lactate dehydrogenase) (Cooper and Hunter, 1981b; Cooper, et a l . , 1983a). FSV P i 4 0 g a g ~ f P s and i ts protease-resistant C-terminal 45 kd fragment phosphorylate enolase and LDH in vitro at the same tyrosine residues as become phosphorylated in FSV-transformed cells (Cooper, et a l . , 1984a), and these substrates display apparent Kms for P140 g a g~ :fP s that are within physiological concentrations. In addition, the association of tyrosine-specific - 97 -protein kinase activity with the cellular receptors for mitogenic hormones such as epidermal growth factor, platelet-derived growth factor and insulin has suggested a normal role for tyrosine phosphory-lation in the induction of cel l division (Ushiro and Cohen, 1980; Ek, et a l . , 1982; Kasuga, et a l . , 1983). If these notions are valid then tyrosine phosphorylation would be expected to exert i ts effect by modulating the enzymatic and biological activities of at least some substrate proteins, as has been fully established in several instances of phosphorylation by serine-specific protein kinases (Cohen, 1982). However, such a function has yet to be demonstrated for any case of reversible tyrosine phosphoryla-tion; indeed substitution of RSV p 6 0 s r c tyrosine-416 with phenyla-lanine destroys the major site of p 6 0 s r c tyrosine phosphorylation but has no obvious effect on src transforming abil ity or kinase ac t i -vity of p 6 0 s r c (Snyder, et a l . , 1983). In the previous chapter I showed that P 1 3 0 g a g _ f P s i s phosphorylated in FSV-transformed cells at three tyrosine residues and at several serine sites. Therefore I have used FSV p i s o ^ ^ ^ P 5 1 as a model substrate to investigate whether tyrosine phosphorylation can in fact modify protein function. The major site of Piso^ g - f P 8 phosphorylation in vivo, as in vitro, is tyrosine-1073 which is located in the C-terminal kinase domain and is homologous to the major site of tyrosine phosphorylation in RSV p 6 0 s r c and Y73 P 9 0 ^ ^ e s (Kitamura, et a l . , 1982; Shibuya and Hanafusa, 1982). In this study I have determined the - 98 -effects of changing tyrosine-1073 of FSV P l 3 0 g a g ~ f p s to a phenyla-lanine residue on the enzymatic activity and biological function of this transforming protein. 4.2 Results 4.2.1 Oligonucleotide-directed Mutagenesis of FSV Oligonucleotide-directed mutagenesis of the FSV genome, cloned into an M13 single-stranded DNA bacteriophage vector has been used to convert tyrosine-1073 within FSV P l 3 0 g a g _ f p s to phenyla-lanine. Since phenylalanine is the closest amino acid in structure to tyrosine, which cannot be phosphorylated, this amino acid substitution should render residue 1073 unavailable for phosphorylation, without perturbing the native conformation of the unphosphorylated protein. The amino acid sequence surrounding tyrosine-1073 and the corresponding nucleotide sequence of the FSV genome are shown in Figure 4.1. A mutagenic oligonucleotide 16 nucleotides long was synthesized such that the TAT codon for tyrosine-1073 is replaced by a TTT codon for phenyla-lanine. However, apart from this single substitution of T for A the sequence of the mutagenic primer is identical to nucleotides 3589-3604 of the FSV genome as defined by Shibuya and Hanafusa (1982) (see Figure 4.1). To provide a template for mutagenesis a 4.7 kbp Sstl restriction endonuclease fragment corresponding to the entire FSV genome in a c i r -cularly permuted form was purified from a XgtWES«^\B vector (Shibuya, - 99 -Figure 4.1 Synthetic oligonucleotides used to mutate the codon for tyrosine-1073 of FSV P 1 3 0 g a g _ f P s . The figure shows (a) the amino acid sequence surrounding tyrosine-1073 and the corresponding wild-type FSV nucleotide sequence (Shibuya and Hanafusa, 1982); (b) the oligonucleotide used to nutate the TAT codon for tyrosine-1073 to a TTT codon for phenylalanine; and (c) the oligonucleotide used to direct the synthesis of a revertant in which the TAT codon is restored. Arrows indicate potential sites of trypsin cleavage in the peptide sequence. y 1073 y ArgGl nGl uGl uAspGl yVal|Tyr[/\l aSerThrGl yGl yMetLys C GGCAGGAGGAGGAT GGT GTCTAT GCCTCCAC GGGGGGCAT GAAG Phe TGGTGTCTTTGCCTCC Tyr TGGTGTCTATGCCTCC - 101 -et a l . , 1982b) and was cloned into the Sstl site of Ml3mpl0 RF DNA (Messing, 1983) (Figure 4.2). Phage containing v iral inserts were propagated in E. col i JMlOl, and those phage ssDNAs with the FSV strand complementary to the oligonucleotide were identified by their abil ity to act as templates for dideoxynucleotide chain termination sequencing reactions in which the mutagenic oligonucleotide served as a sequencing primer. These reactions also showed that the mutagenic oligonucleotide only hybridized stably to the expected site within the FSV insert. A representative phage (M13mplOFSV-8) was mutagenized by a two-primer method (Zoller and Smith, 1984) in which the mutagenic oligonucleotide and a second oligonucleotide complementary to an M13 sequence (the universal sequencing primer) were both annealed to M13mplOFSV-8 phage ssDNA. E. col i DNA polymerase I (large fragment) was used to extend the primers in the presence of the four deoxynucleoside triphosphates and T4 DNA ligase. This DNA was introduced into competent bacteria, and ssDNAs isolated from the resulting plaque purified phage were screened using the mutagenic oligonucleotide as a hybridization probe under conditions of increasing stringency to identify phage with the desired mutation in their FSV inserts. Of 24 different phage DNAs tested, 22 bound the probe at 40°C but not at 50°C and were presumed to s t i l l contain a wild-type FSV sequence with a single base mismatch to the mutagenic oligonucleotide. Two phage DNAs remained hybridized to the probe at 50°C (Figure 4.2), and were presumed to be mutants. DNAs of a putative wtFSV phage (M13mpl0wtFSV) and a mutant phage (M13mplOFSV-F(1073)) were sequenced in the region encompassing the - 102 -Figure 4.2 Strategy for the oligonucleotide-directed mutagenesis of FSV. The diagram illustrates the method by which the 4.7 kbp FSV genome was subcloned from X-FSV-2 into the Sstl site of M13mpl0 and the TAT codon for tyrosine-1073 of P130 g a g ~ f P s mutated to TTT using the oligonu-cleotide displayed in Figure 4.1(b). Putative mutant phage ssDNAs were dotted onto nitrocellulose and screened for their abi l i t ies to hybridize with the 32 P-labelled mutagenic oligonucleotide under condi-tions of increasing temperature. - 103 -ISOLATE 4.7 kbp FSV INSERT OF A-FSV-2 DNA y CLONE INTO S s t l SITE OF M13mpl0 RF DNA gag fps LTR ISOLATE PHAGE WITH FSV INSERT IN DESIRED ORIENTATION OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS T SCREEN RESULTING PHAGE DNAs BY DOT-BLOT HYBRIDIZATION WITH THE MUTAGENIC OLIGONUCLEOTIDE AS A LABELED PROBE - 104 -mutation site (Figure 4.3). The two sequences are identical except that adenine contained within the TAT codon for tyrosine-1073 of wtFSV DNA is mutated to a thymine in FSV-F(1073) DNA yielding a phenylalanine codon. 4.2.2 Synthesis of a Revertant FSV In order to prove that any biological or biochemical dif fer -ences between the p l 3 0 gag- fps proteins encoded by wtFSV and FSV-F(1073) resulted from the conversion of tyrosine-1073 to phenyla-lanine and not from a spontaneous mutation at another unrelated site I reverted the mutant DNA back to the wild-type. To do this I synthe-sized an oligonucleotide 16 long identical to the original wtFSV sequence between nucleotides 3589-3604 (see Figure 4.1) and used this as a mutagenic oligonucleotide with M13mplOFSV-F(1073) phage ssDNA as the template for mutagenesis. The same protocol described above was used to isolate an M13 phage in which the TTT codon for phenylalanine-1073 in the mutant FSV-F(1073) insert has been mutated back to a TAT codon for tyrosine. The nucleotide sequence of this phage Ml3mpl0FSV-Y(1073) is identical to wtFSV in the region of interest (Figure 4.3). Any biological activity of wtFSV that is altered in the mutant FSV-Y(1073) but restored to wild-type function in the revertant FSV-Y(1073) must vary solely in response to the amino acid changes at residue 1073 in PISO^S"^ 8 . - 105 -Figure 4.3 Partial DNA sequencing of wtFSV, mutant FSV-F(1073) and revertant FSV-Y(1073) M13mpl0 inserts. The nucleotide sequences encompassing the codon for tyrosine-1073 of wtFSV and the mutation sites of FSV-F(1073) and FSV-Y(1073) DNAs were determined by the dideoxynucleo-tide chain termination method. The codons for amino acid 1073 are arrowed in each case. wtFSV FSV-F(1073) FSV-Y(1073) WILD TYPE MUTANT REVERTANT - 107 -4.2.3 Transforming Activity of Wild-type, Mutant and Revertant FSV  DNAs The wtFSV, FSV-F(1073) and FSV-Y(1073) sequences from the appropriate M13 vectors were assayed for their abi l i t ies to transform rat-2 cel ls . The 4.7 kbp FSV inserts were purified by agarose gel electrophoresis following Sstl digestion of RF DNAs isolated from phage-infected cells. FSV inserts were electroeluted from the gels, ligated to reconstruct intact FSV genomes (Shibuya, et a l . , 1982b) and introduced into rat-2 cells by the calcium phosphate co-precipitation transfection technique (Graham and Van der Eb, 1973). Within 8 days foci of transformed cells with a round, refractile morphology were apparent in dishes of rat-2 cells transfected with wtFSV DNA or with revertant FSV-Y(1073) DNA (Figure 4.4). At two weeks post-transfection wtFSV DNA gave 50-100 foci/ug DNA/5 x 106 cel ls , and by three weeks approximately five times more foci were visible, though many of these may have arisen by secondary spread of transformed cel ls . In con-trast, rat-2 cells transfected with FSV-F(1073) mutant DNA showed no evident morphological change unti l at least 30 days post-transfection, when some foci of transformed cells with a fusiform morphology became visible (Figure 4.4). If the cells tranfected with FSV-F(1073) mutant DNA were maintained for 2-3 months the i n i t i a l foci became prominent and acquired a more pronounced, round morphology. Thus, the FSV-F(1073) DNA encoding phenylalanine in place of tyrosine-1073 at position 1073 is s t i l l able to induce transformation of rat-2 cel ls , - 108 -Figure 4.4 Transformation of rat-2 cells following transfection with FSV DNAs. Rat-2 cells were transfected with wild-type, mutant or revertant FSV DNAs and examined for the appearance of foci of transformed cells: Normal rat-2 cells (A); a typical focus 15 days after transfection with wtFSV DNA (B); a focus 44 days after tranfection with mutant FSV-F(1073) DNA (C); a focus 15 days after transfection with revertant FSV-Y(1073) DNA (D). Cell lines of wtFSV-transformed rat-2 cells (E) and FSV-F(1073)-transformed cells (F) derived from such foci are shown. The appearance of colonies 3 weeks after seeding in soft agar are shown for normal rat-2 cells (G); wtFSV-transformed cells (H); FSV-F(1073)-transformed cells (I); and FSV-Y(1073)-transformed cells (J). FSV FSV 8 FOCI CELL LINES COLONIES - 110 -but does so with a much longer latent period than wtFSV or revertant FSV DNAs. Foci of transformed rat-2 cells which appeared following transfection of wtFSV, mutant FSV-F(1073) or revertant FSV-Y(1073) DNAs were picked, subcloned and expanded in mass culture (Figure 4.4). A l l such morphologically transformed cel l lines, including those induced by FSV-F(1073), formed large colonies in soft agar (Figure 4.4) unlike the normal rat-2 cells (Figure 4.4G). To determine whether the mutant FSV-F(1073) transformed rat cells could induce tumors in vivo, wtFSV, mutant FSV-F(1073), revertant FSV-Y(1073) or rat-2 cells were injected subcutaneously, at a cel l concentration of 1.0 x 10^ cells per .1 ml into the back of the neck of 4-5 week old female Fischer rats, in tr ipl icate. Approximately three weeks post-injection tumors appeared at the site of injection in rats that had been injected with wtFSV or revertant transformed cel ls. However, tumors were not obvious until 6-8 weeks post-injection with rats injected with mutant FSV-F(1073) transformed cells. In addition, tumors appeared at the site of injection in some animals that had been injected with normal rat-2 cel ls , but this occurred only after 5 months following injection and probably reflects the properties of this immortalized, continuous cel l l ine. These results are an average of three independent experiments and they support the enhanced latent period of transformation seen with the mutant FSV-F(1073) in vitro. - I l l -Tumors from these animals were excised, grown in tissue culture and subsequently assayed for the expression and activity of P 1 3 0 g a g - f p s as described below. Tumors induced by mutant FSV-F(1073) transformed cells contained cells which expressed P 1 3 0 g a g - f p s characteristic of the mutant protein, such as enhanced mobility in SDS gels and reduced in vitro tyrosine specific kinase activity compared to of the wild-type or revertant transforming proteins (see below). 4.2.4 Expression, Structure and In Vitro Kinase Activities of Wild- type and Mutant P 1 3 0 g a g ~ f p s Proteins Equal numbers of cells transformed with wtFSV, FSV-F(1073) or with FSV-Y(1073) were labelled with [35S]methionine for 16 hours, lysed and immunoprecipitated with a n t i - p l 9 g a g serum. Equivalent amounts of labelled P 1 3 0 g a g _ f p s were immunoprecipitated from each line of transformed cells (Figure 4.5). Pulse-chase experiments have shown that the wtFSV and FSV-F(1073) P 1 3 0 g a g - f p s proteins have approximately the same turnover rates (data not shown) and o r [ S]methionine-labelling can therefore be used as a relative measure of P 1 3 0 g a g ~ f p s levels. The mobilities of the wtFSV and revertant proteins were identical, demonstrating that the mutagenesis procedures had not grossly affected the FSV coding sequence. Mutant PISO 5 3 ^ - ^ 8 encoded by FSV-F(1073) migrated slightly more rapidly than the wild-type or revertant proteins, possibly as a result of decreased phosphorylation. In a parallel experiment wild-type, mutant or revertant P130 g a g ~^ p s proteins were immunoprecipitated from - 112 -Figure 4.5 Analysis of wild-type, mutant and revertant FSV-trans-formed rat-2 cells for FSV P 1 3 0 g a g ~ f p s synthesis and tyrosine-specific protein kinase activity. Normal and transformed rat-2 cells were labelled for 16 hours with 100 uCi [ S]methionine, immunoprecipitated with a n t i - p l 9 g a g serum and examined by gel electro-phoresis to identify p i 3 0 g a g _ f P s (lanes A-E). Cells from duplicate unlabelled cultures were immunopre-cipitated in identical fashion and then incubated with 5 ug of acid-denatured enolase in the presence of 10 mM MnCl2 and 2.5 uCi ["^-32P]ATP for 15 minutes at 30°C (lanes F-J). Samples were anlaysed by electro-phoresis through a 7.5% SDS-polyacrylamide gel and auto-radiography. Immunoprecipitates were from normal rat-2 cells (lanes C and H) or from rat-2 cells transformed with wtFSV (A and F); with FSV-F(1073) (B, D, G, and I); or with FSV-Y(1073) (E and J). - 114 -equal numbers of unlabelled c e l l s and the immune complexes incubated qp with MnCl 2, [ J - P]ATP and soluble, denatured rabbit muscle enolase (Figure 4.5). In t h i s reaction wtFSV PISO 8 3- 6"^ 8 i s auto-phosphorylated and also phosphorylates enolase at a single tyrosine i d e n t i c a l to that phosphorylated i n FSV-transformed chicken embryo fi b r o b l a s t s (Cooper, et a l . , 1984a). Mutant FSV-F(1073) PISO^S"* 1* 3 c l e a r l y functions i n v i t r o as a protein kinase but i s apparently less active i n phosphorylating enolase than wtFSV or FSV-Y(1073) p 1 3 0gag-fps. The extent of enolase phosphorylation i n each case was normalized to the amount of immunoprecipated P130 g a g~^P 8 by qp determining the P or S cpm i n the relevant bands (Table 4.1). The mutant protein i s approximately f i v e - f o l d less active i n enolase phosphorylation than the wild-type or revertant proteins when assayed i n t h i s way. The considerable decrease i n r a d i o l a b e l l i n g of FSV-F(1073) p l 3 0 g a g - f p s i t s e l f i n the immune complex kinase reaction (Figure 4.5, Table 4.1) would be expected i f the major s i t e of i n v i t r o t yro-sine autophosphorylation had been destroyed by substituting tyrosine-1073 with phenylalanine. C-terminal fragments of i n vitro-phosphory-lated mutant p l 3 0 g a g - f p s generated by cleavage with V8 protease were poorly phosphorylated compared with corresponding fragments of the wtFSV protein (data not shown). To confirm that the expected amino acid substitutions had been introduced at residue 1073 and to define the effect of these substitutions on phosphorylation of the mutant and - 115 -TABLE 4.1 Quantitation of the Kinase Activities of P130 g a g"" fP s  Proteins Encoded by Wild-Type, Mutant and Revertant FSV DNAs Relative Phosphorylation8- in vitro by P l 3 0 g a g ~ f P s Source of Enolase pisoga-g-fps PISOgag-fPS (autophosphorylation) wtFSV 7.5 b 9.7° 0.6 d FSV-F(1073) 1.4 2.2 0.1 FSV-Y(1073) 6.0 ND 0.6 a Immune complex kinase reactions were performed as shown in Figure 4.5 following immunoprecipitation of P 1 3 0 g a g - f p s from rat-2 cells transformed by wtFSV, FSV-F(1073) or FSY-Y(1073). The extent of enolase phosphorylation or P 1 3 0 g a g - f P s autophos-phorylation was measured by counting appropriate dried gel slices for 3 2 P . The amount of P 1 3 0 g a g _ f P s in each immunopre-cipitate was estimated by labelling duplicate dishes of cells with [35S]methionine, immune-precipitating PISO^^^P 8 and counting the gel-purified protein. The values shown are the ratios of P cpm incorporated into the substrate to S cpm in P130 g a g ~ f P s . b,c Values are from two separate experiments. ND, not done. d Mean of two separate experiments. - 116 -revertant proteins tryptic digests of wtFSV, FSV-F(1073) and FSV-Y(1073) p l 3 0 gag-fps j w n i c n had been radiolabelled in vivo with 3 2 P i or [ S]methionine or autophosphorylated in vitro using qp [ 5 - P]ATP were analysed. Analysis of the tryptic peptide con-taining tyrosine-1073 is complicated by the location of a glutamine residue at i t s N-terminus which, as described in Chapter 3, can be modified during the mapping procedure yielding three separately migrat-ing peptide species. A tryptic phosphopeptide map of P l 3 0 g a g ~ ^ p s autophosphorylated in an immune complex kinase reaction (Figure 4.6, map C) reveals five spots of which three (previously designated 3a, 3b and 3c) are a l l derived from the same tryptic peptide thought to con-tain tyrosine-1073 whereas the remaining two correspond to less promi-nent sites of phosphorylation (Chapter 3). The tryptic peptide encom-passing tyrosine-1073 also possesses a methionine residue, and a tryp-t i c peptide map of [35S]methionine-labelled wtFSV pisoS 8- 6"* 1 5 8 should therefore contain radiolabelled peptides corresponding both to the phosphorylated species 3a-3c and their non-phosphorylated counter-parts in a ratio dependent on the stoichiometry of phosphorylation. A map of mutant FSV-F(1073) PISOS 3- 6""* 1 5 8 labelled by in vitro phosphorylation (Figure 4.6, map F) is entirely lacking spots 3a-3c but retains the two minor tryptic phosphopeptides, confirming that 3a-3c represents phosphorylation of tyrosine-1073. In contrast, the tryptic phosphopeptide map of revertant p 1 3 0gag-fps phosphory-lated in an immune complex is identical to that of wtFSV - 117 -Figure 4.6 Tryptic peptide analysis of P 1 3 0 gag-fps encoded by wild-type, mutant and revertant FSVs. p l 3 0 gag- fps isolated from transformed rat-2 cells labelled in vivo with [35S]methionine (A, D, G) or with 3 2 P -orthophosphate (B, E, H) or labelled with 3 2 P in vitro by autophosphorylation in immune complex kinase reactions (C, F, I) was subjected to 2-dimensional tryptic peptide mapping. p l 3 0 gag- fps was isolated from rat-2 cells transformed with wtFSV (A, B, C) or with FSV-F(1073) (D, E, F) or with FSV-Y(1073) (G, H, I). tsFSV P 1 4 0 g a g _ f P s was isolated from tsFSV(FAV)-infected CEFs which were labelled with [ S]methionine (J, K). CEFs were maintained at 37°C' (J) or at 41.5°C (K). [35S]methionine-32 labelled proteins and P-labelled proteins were oxidized, digested with trypsin and separated in two dimensions in identical fashion. Origins are marked with an 'VD". Electrophoresis at pH 2.1 is displayed from left to right; anodes are to the left and cathodes are to the right. Chromatography in the second dimen-sion was from bottom to top. The identities of unmarked tryptic peptides can be found in Pawson, et a l . (1981) or Chapter 3. The double spot to the right of phospho-peptide 4 in maps E and F and the spot above 3c in map F are variable and have also been observed in wtFSV Pl 3 0gag-fps (see Chapter 3). - 118 -35 w I L D T Y P E S MET WTFSV A WTFSV 00 30 P IN VIVO P IN VITRO B WTFSV M u T A N T R E V E R T A N T T E M P E R A T U R E S E N S I T I V E -3c y 3bv 3bp-¥ FSV F(1073) • 3 c . 3b • FSV-Y(1073) 3c 3b" D FSV~F(1073) G FSV Y(1073) 3b^ . *^3b p tsFSV 37°C * 3a 3ap 4 • J i . D - V % e 3b p tsFSV 41.5°C K 3c' 3b • • * 3 c p y 1 » ^ # 3 b * £ FSV F(1073) I H FSV-Y(1073) 3b p y - 119 -p l 3 0 gag- fps ( F i g u r e 4 . 6 ) ^ j.). These findings shew that tyrosine-1073 is indeed the major site of wtFSV piSO^S^P 8 auto-phosphorylation, and that this phosphorylation site is lost in the mutant protein but restored in revertant P 1 3 0 g a g ~ f p s . To under-take a more detailed structural analysis of the pro-teins, and to identify the mutant phenylalanine-1073-containing tryptic peptide I used in vivo labelling with [35S]methionine. wtFSV p l 3 0 gag- fps contains [35S]methionine-labelled tryptic peptide species which co-migrate with tryptic phosphopeptides 3a, 3b and 3c of 3 2 P-labelled PISOS3"6"^8 (Figure 4.6, Map A). Presumably they correspond to the phosphorylated form of the tyrosine-1073-containing tryptic peptide with an unmodified (3c) or modified (3a, 3b) N-terminal glutamine, and are designated 3aP~v, 3bP~v and 3cP~v to indicate the presence of a phosphotyrosine. To confirm the identity of these methionine-containing tryptic peptides I have used a variant of FSV which is temperature-sensitive for transformation (tsFSV L-5) and which encodes a P 1 4 0 g a g - f P 8 protein which is highly phosphorylated at tyrosine in cells maintained at the permissive temperature for transformation (37°C) but only poorly phosphorylated at tyrosine at the nonpermissive temperature (41.5°C) (Pawson, et a l . , 1980). tsFSV P l 4 0 g a g ~ f P s from infected chicken embryo fibroblasts grown at 37 °C contains [ S]methionine-labelled tryptic peptides 3aP~ v-3CP~ v in similar yield to wtFSV P130^^~fPs (Figure 4.6, map J), but these peptides are barely detectable in p14Qme-fps f r o m ^fected cells maintained at 41.5°C (Figure 4.6, - 120 -map K), as would be expected i f they contained a reversibly phosphoryl-ated tyrosine residue. I then analysed a tryptic digest of methionine-labelled mutant FSV-F(1073) P 1 3 0 g a g _ f p s (Figure 4.6, map D) and could detect no spots corresponding to phosphorylated 3 a p - y -3 Cp-y, consistent with predicted substitution of phenylalanine for tyrosine-1073. In addition to the absence of 3aP~y-3cp~'y, mutant P 1 3 0 g a g ~ f p s lacks two further wild type peptides (desig-nated 3b y , 3c y on maps of wild-type and revertant P 1 3 0 g a g - f p s ) but has acquired two novel peptides (designated 3b f and 3c f ) . The 3b y and 3c y wild-type peptides missing from the digest of mutant P 1 3 0 g a g - f p s migrate as i f they were less negatively charged and more hydrophobic than the phosphorylated Sb15-"57 and 30^^. My interpretation of these data is that wild-type peptides 3by and 3c y correspond to the non-phosphorylated forms of the tyrosine-1073-con-f f taming tryptic peptide and that the mutant peptides 3b and 3b represent the same tryptic peptide but with phenylalanine at residue 1073. Mutant 3b and 3c peptides migrate very similarly to 3b y and 3c y , but move more rapidly in the chromatographic dimension of the 2D map consistent with the increased hydrophobicity expected from substituting phenylalanine for tyrosine. With these exceptions, the methionine containing tryptic peptides of mutant P 1 3 0 g a g ~ f p s are identical to those of wtFSV P 1 3 0 g a g ~ f p s . The tryptic peptide map of [35S]methionine-labelled revertant piaogag-fps (Figure 4.6, map G) has a similar pattern to that of the wild-type protein, indicating that the differences between wild-type and mutant tryptic peptides result solely from the substitution at residue 1073. - 121 -These data a l l support the assertions that tyrosine-1073 is the major site of P l 3 0 g a g " " f p s tyrosine phosphorylation, that this residue has been substituted with phenylalanine and is no longer phos-phorylated in FSV-F(1073), and has been restored to tyrosine and i s once more phosphorylated in FSV-Y(1073). 4.2.5 Tyrosine Phosphorylation in wtFSV and FSV-F(1073)-transformed  Cells wtFSV P130^^~fPs i s phosphorylated at three tyrosine residues in FSV-transformed cel ls . A minor and variable site Of tyro-sine phosphorylation l ies within the N-terminal gag region (peptide 1) and two phosphotyrosine. sites are located in the fps-encoded region: the major site at tyrosine-1073 (peptides 3a-3c) and an additional fps site contained within peptide 4 (Figure 4.6, map B). Several serine residues are also phosphorylated. wtFSV and FSV-F(1073) P 1 3 0 gag-fps proteins were isolated from 3 2 P-labelled trans-formed rat-2 cells and analysed for phosphoamino acid content and by tryptic phosphopeptide mapping. The mutant protein contained approxi-mately two-fold less phosphotyrosine, relative to i ts total phospho-amino acids, than the wild-type protein (Figure 4.7, Table 4.2) and this is explained by the absence of tryptic phosphopeptides 3a-3c from digests of in vivo-labelled FSV-F(1073) P 1 3 0 g a g ~ f p s (Figure 4.6, map E). The mutant protein is s t i l l phosphorylated in vivo at the second fps tyrosine site (peptide 4) and at the minor gag tyrosine site - 122 -Figure 4.7 Phosphoamino acid analysis of P I S O ^ ^ P 8 . Rat-2 cells transformed with wtFSV (A) or mutant FSV-F(1073) qp (B) were labelled with P-orthophosphate for 4 hours, lysed, immunoprecipitated with a n t i - p l 9 g a g serum and the immunoprecipitates analysed by gel elec-trophoresis. 3 2 P-labelled p i 3 0 g a g - f P s as recovered from the gels, acid-hydrolyzed and analysed for phosphoamino acids by two-dimensional electro-phoresis at pH 1.9 and pH 3.5 followed by autoradio-graphy. The positions of marker phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) as revealed by ninhydrin staining are indicated. - 123 -- 124 -TABLE 4.2  Phosphoamino Acid Analysis of P 1 3 0 gag-fps and Total Cellular Protein PISO^^PS Total Cell Protein Phosphoamino a acid wtFSV FSV-F(1073) rat-2 wtFSV FSV-F(1073)b p-serine 65° 82 95 90 91 p-threonine 13 8 5 9.7 8 p-tyrosine 22 10 .07 .39 .27 Radioactivity in individual phosphoamino acids of P130gag~-*-Ps isolated from 3 2 P-labelled cells or of total cellular 3 2 P -labelled protein was determined by scinti l lation counting of aspirated thin-layer cellulose spots following electrophoretic separation of acid hydrolysates (Figures 4.7 and 4.8). a wtFSV-transformed rat-2 cel ls . b FSV-F(1073)-transformed rat-2 cells. c Values for each phosphoamino acid are expressed as a percentage of total phosphoamino acids for that sample. - 125 -(peptide 1), though this latter phosphopeptide is not apparent in the map shown in Figure 4.6. In addition FSV-F(1073) P 1 3 0 g a g - f P s i s phosphorylated at the same serine sites in vivo as wtFSV p l 3 0 gag- fps as judged by the migration of phosphoserine-containing tryptic peptides. Thus the loss of tyrosine-1073 does not appear to qualitatively affect the phosphorylation of other tyrosine or serine residues within P 1 3 0 g a g - f p s . A tryptic phosphopeptide map of revertant FSV-Y(1073) PISO^^P* 5 labelled in vivo with 32 Pi (Figure 4.6, map H) once more contained peptides 3a-3c. In a series of independent tryptic digests the only consistent difference between the tryptic phosphopeptides of wtFSV, FSV-F(1073) and FSV-Y(1073) P l 3 0 g a g ~ f P s was the absence of 3a-3c from the mutant protein. Transformation by FSV induces an increase in total cel l phos-photyrosine resulting from phosphorylation of a variety of cellular proteins at tyrosine (Pawson, et a l . , 1980; Cooper and Hunter, 1983b). Normal rat-2 cells and the rat-2 lines transformed by wtFSV or the mutant FSV-F(1073) were labelled with J*P-orthophosphate and analysed for whole cel l phosphoamino acid content (Figure 4.8; Table 4.2). In one experiment wtFSV-transformed cells had 5.6-fold more phos-photyrosine than normal rat-2 cel ls , and rat-2 cells transformed by the mutant FSV-F(1073) showed a 3.8-fold elevation of phosphotyrosine. FSV-F(1073) can therefore s t i l l induce the phosphorylation of cellular proteins at tyrosine, indicating that the mutant protein is active as a kinase in vivo. - 126 -Figure 4.8 Whole cel l phosphoamino acid analysis. Normal rat-2 cells (A) or rat-2 cells transformed by wtFSV (B) or mutant FSV-F(1073) (C) were labelled for 5 hours with 32 P i and total cellular protein was extracted, acid-hydrolyzed and separated in two dimensions by electrophoresis at pH 1.9 and pH 3.5. The mobilities of marker phosphoamino acids are phosphoserine (S), phos-phothreonine (T) and phosphotyrosine (Y) as identifed by ninhydrin staining. - 127 -4 0 - 128 -4.3 Discussion The substitution of phenylalanine for tyrosine at residue 1073 of ^ 1 3 0 ^ ^ ^ does not abolish the abil i ty of FSV to trans-form rat-2 cel ls. However there is a marked delay in the appearance of foci in rat-2 cells transfected with the mutant compared with those transfected with wtFSV. The long latent period of focus formation of FSV-F(1073) must reflect the loss of tyrosine-1073 since the revertant FSV-Y(1073) is rapidly transforming, and this suggests that the mutant protein is functionally altered as a result of the conversion of tyrosine-1073 to phenylalanine. The tyrosine-specific kinase activity of the mutant protein, as measured by in vitro phosphorylation of rabbit muscle enolase, is reduced five-fold compared with wtFSV or revertant PlSOgag -^ 8 . It i s reasonable to propose that the reduced kinase activity of the mutant FSV-F(1073) p i 3 0 g a g _ f p s results from i ts inability to become phosphorylated at residue 1073 and that this reduced enzymatic activity is responsible for the ine f f i -ciency of the mutant FSV-F(1073) in inducing transformation of rat-2 cel ls . This conclusion rests on the assumption that phenylalanine at position 1073 does not disturb the conformation and activity of P 1 3 0 g a g - f p s . The functional differences between the mutant and wild-type P 1 3 0 gag-fps can be attributed to the substitution at residue 1073, since the wtFSV and revertant FSV-Y(1073) DNAs and P 1 3 0 g a g ~ f p s proteins are identical in a l l aspects of structure and biological - 129 -activity tested. These findings also demonstrate the stability of the 4.7 kbp FSV genome in the M13mpl0 vector through two cycles of oligonucleotide-directed mutagenesis. There are several examples of serine-specific protein kinases whose activities are enhanced by their own phosphorylation at serine; the stimulation of phosphorylase kinase activity by phosphorylation of i t s B-subunit by cAMP-dependent protein kinase is well-documented (Cohen, 1982). There is some circumstantial evidence that the activity of tyrosine-specific protein kinases may be stimulated by their own phosphorylation at tyrosine (see section 1.10.1). Preincubation of purified insulin receptor or RSV p 6 0 s r c under conditions promoting their autophosphorylation enhances their subsequent abil ity to phos-phorylate exogenous substrates (Rosen, et a l , 1983; Purchio, et a l . , 1983). The 98 kd product of the normal avian cellular fps gene (NCP98) is expressed in bone marrow cells (Shibuya, et a l . , 1982a). NCP98 possesses in vitro autophosphorylating tyrosine-specific kinase ac t i -vity but is not detectably phosphorylated at tyrosine in cultured cells (Mathey-Prevot, et a l . , 1982). It is possible that the phosphorylation of P130 g a g ~^ p s in vivo represents a unique feature of the v i ral transforming protein which fortuitously leads to increased kinase ac t i -vity. Perhaps, NCP98 becomes transiently phosphorylated at tyrosine in the animal in response to a specific environmental signal (which is lost in tissue culture) resulting in turn in a short-lived stimulation of NCP98 activity and function at a defined stage of hematophoiesis. - 130 -The decreased kinase activity and inefficient transforming function of the FSV-F(1073) mutant protein indicate that the high level of wtFSV P 1 3 0 g a g _ f p s phosphorylation at tyrosine-1073 results in constitutive enzymatic activation which in turn is important in the induction of unregulated cellular proliferation. The basis for the long latency of transformation by FSV-F(1073) is unknown. Recombination with c-fps resulting in restoration of the codon for tyrosine-1073 does not seem likely since a l l of the P I S O 8 3 6 - ^ 8 detectable in FSV-F( 1073)-transformed cells retains phenylalanine at residue 1073 as judged by peptide mapping and migrates more rapidly than wtFSV P 1 3 0 g a g - f p s in SDS-polyacrylamide gel electrophoresis. It is possible that the latent period reflects the requirement for activation of a cellular gene that co-operates with mutant PISO6 9"6 -"^8 in inducing the transformed phenotype, as suggested for the transformation of B-lymphocytes by Abelson murine leukemia virus (Ab-MuLV) whose transforming protein is also a tyrosinespecific protein kinase (Whitlock, et a l . , 1983). Even though the molecular basis of the enhanced latent period of transformation associated with the mutant FSV-F(1073) is unknown, i t must reflect the oncogenic status of the mutant transformed cells since these cells also induce tumors in animals only after a longer period of time than that required by the wtFSV or revertant FSV-Y(1073) transformed cel ls . - 131 -The phenotypic consequences of changing tyrosine-1073 of FSV P 1 3 0 g a g - f p s are much more dramatic than those observed for corresponding mutants of RSV src. The major site of RSV p 6 0 s r c tyrosine phosphorylation, tyrosine-416, which is homologous with plSOgag-fps tyrosine-1073 has recently been changed to phenylalanine (Snyder, et a l . , 1983) or deleted (Cross and Hanafusa, 1983). Cross and Hanafusa reported a slightly longer latent period for transformation, decreased tumorigenicity and possible reduction in kinase activity of p 6 0 s r c for the pSR-XDT10-l deletion mutant of RSV src. However, since this construction contained a small deletion and substitution these biological effects could not be certainly ascribed to the loss of tyrosine-416. Synder, et a l . , (1983) have observed that the substitution of p 6 0 s r c tyrosine-416 with phenylalanine has no apparent effect on transformation of mouse fibroblasts in culture or p 6 0 s r c kinase activity in vitro, in contrast to my results with FSV. Although further investigations reveal that this src mutant is very poorly tumorigenic suggesting that i t has indeed sustained a functional lesion (Snyder and Bishop, 1984). These results do not exclude the possibility that some ac t i -vity of FSV PISO^3^ -^8 1 other than tyrosine phosphorylation is involved in i ts transforming abi l i ty . However, these data strongly suggest that tyrosine phosphorylation can modulate enzymatic activity, and therefore strengthen the case for involvement of tyrosine phosphorylation in transformation by FSV and similar viruses. The - 132 -apparent effect of tyrosine-1073 phosphorylation on FSV P 1 3 0 g a g _ f p s activity is consistent with the general concept that reversible protein phosphorylation at a single site can enhance or depress activity by converting an enzyme from one structural form to another, but does not usually act as an absolute functional activator or inhibitor. Apparently phosphorylation of the FSV transforming protein at tyrosine is an important factor in regulating i ts oncogenic action. - 133 -CHAPTER 5 5.0 The Protein Kinase Activity of FSV PISOS3-5"^5 Shews a Strict  Specificity for Tyrosine Residues. 5.1 Introduction A number of oncogenic viruses encode transforming proteins with protein kinase activities specific for tyrosine residues (Bishop and Varmus, 1982; Cooper and Hunter, 1983b). Tyrosine phosphorylation i s a relatively rare event and appears to be correlated with cellular transformation. As a result, the basis on which oncogenic tyrosine protein kinases select their substrates is of interest as i t l ikely relates to the mechanism of viral transformation. Other kinases, such as the cAMP-dependent protein kinases appear to recognize their substrates ty the primary sequence around the phosphorylated residue. Studies using synthetic peptide substrates show that the serines phosphorylated the cAMP-dependent protein kinases usually have one or two basic residues on the N-terminal side of the phosphorylated amino acid (see section 1.9). However, a number of other studies have indicated that these enzymes must also recognize a specific secondary structure in their substrates (Shenolikar and Cohen, 1978; Zetterquist and Ragnarsson, 1982). - 134 -Synthetic peptides have also been used to determine the sub-strate specificity of tyrosine protein kinases. The presence of acidic residues on the N-terminal side of the phosphorylatable tyrosine appears to be a factor in substrate recognition by several tyrosine protein kinases (Patschinsky, et a l . , 1982; Pike, et a l . , 1982; Hunter, 1982). However, some of the sites phosphorylated in proteins do not have acidic residues on the N-terminal side of the phosphorylated tyro-sine residue (Gallis, et a l . , 1983; Guild, et a l . , 1983; Cooper, et a l . , 1984a). In addition, the angiotensin peptides are reasonably good substrates for a number of tyrosine protein kinases, even though none of them have a large number of acidic residues in their sequences (Wong and Goldberg, 1983b). Therefore i t appears that factors other than the presence of acidic residues contribute to the recognition of phosphorylation by tyrosine kinases. As in the case of the cyclic nucleotide dependent protein kinases, secondary structure is probably also an important recognition factor. In any case, the studies using synthetic peptides as substrates suggest that tyrosine kinases have a str ict specificity for phosphorylating tyrosine residues (Pike, et a l . , 1982; Hunter, 1982; Wong and Goldberg, 1983b). In contrast, there is evidence to suggest that some of the tyrosine kinases may phosphorylate non-protein substrates such as glycerol (Richert, 1983), diacylglycerol and phosphatidylinositol (Sugimoto, et a l . , 1984; Macara, et a l . , 1984), which raises questions as to their substrate specificity in general, and the physiological - 135 -relevance of tyrosine phosphorylation in particular. However, the high concentrations of glycerol required to detect the phosphorylated pro-duct makes i t unlikely that the enzyme functions as a glycerol kinase in vivo. In addition, the purity of the enzyme preparations used to demonstrate l ip id phosphorylation are somewhat questionable. I have investigated the specificity of the protein kinase activity intrinsic to FSV P 1 3 0 g a g - f p s by using site-directed muta-genesis to change the codon for tyrosine-1073 to those for the other commonly phosphorylated hydroxyamino acids serine and threonine. This approach has several advantages over the use of synthetic peptides to define the protein kinase recognition site. Most important, the pro-tein containing the altered target site may be expressed in intact cel ls , which allows the specificity and dynamics of phosphorylaton to be examined in vivo. As a consequence, such studies wi l l not only allow the primary structure of the enzyme recognition site to be considered, but may also allow the contributions of secondary and tertiary structure to be evaluated. 5.2 Results 5.2.1 Oligonucleotide-directed Mutagenesis of FSV The 4.7 kilobase FSV genome was subcloned from \ -FSV-2 (Shibuya, et a l . , 1982b) into the M13mpl0 bacteriophage vector to - 136 -provide a template for oligonucleotide-directed mutagenesis as pre-viously described (section 4.2.1). The oligonucleotides used to change the TAT codon for tyrosine-1073 within P 1 3 0 g a g ~ f p s to those for serine, threonine or glycine residues are shown in Figure 5.1. The procedures for mutagenesis, bacterial transformation, isolation of phage and the hybridization technique used to screen for mutant phage were exactly as described in section 4.2.1 of the preceding chapter. DNAs isolated from the putative mutant phage were sequenced in the region encompassing the mutation site to confirm the desired mutations (Figure 5.2). The sequences are a l l identical except for the TAT codon for tyrosine-1073 which has been changed to a TCT codon for serine in [FSV-S(1073)], a ACT codon for threonine in [FSV-T(1073)] and a GGT codon for glycine in [FSV-G(1073)J. To ensure that any functional changes associated with the mutants were due to the specifically induced mutations and not the product of random second-site mutations that could have occurred during the mutagenesis procedure, the mutated genomes were each reverted using an oligonucleotide encoding the wild-type sequence (see Figure 5.1). The same protocol described above was used to revert the mutant genomes. The nucleotide sequences of these revertant phage were shown to be identical to the wtFSV in the region of interest (data not shown). Therefore any biological activity of wtFSV that is altered in any of the mutants but restored to wild-type function in the revertant FSV-Y(1073) genomes must be due solely to the amino acid change at residue 1073 in PlSQgag-fps^ Figure 5.1: The synthetic oligonucleotides used to mutate the codon for tyrosine-1073 of FSV P 1 3 0 g a g - f p s . Oligonucleotides 16 long were designed to alter the codon for residue 1073 by one or two nucleotides to code for a serine, threonine or glycine as indicated. The mutagenic oligonucleotide encoding the wild type codon TAT was used to revert each of the mutants to FSV-Y(1073). The figure displays amino acids 1066-1080 of wtFSV P 1 3 0 g a g - f P s and their encoding nucleotide sequence. .... ArgGlnGluGluAspGlyValTyrAlaSerThrGlyGlyMetLys .... C .... CGGCAGGAGGAGGATGGTGTCTATGCCTCCACGGGGGGCATGAAG .... 3' WILD TYPE FSV 5* - TGGTGTCTCTGCCTCC - 3' Ser FSV-S(1073) ACT Thr FSV-T(1073) GGT Gly FSV-G(1073) TAT Tyr FSV-Y(1073) - 138 -Figure 5.2: Partial DNA sequencing of wtFSV and mutant [FSV-S(1073); FSV-T(1073); FSV-G(1073)] M13mpl0 inserts. The nucleo-tide sequences encompassing the codon for tyrosine-1073 of wt FSV and the mutation sites of FSV-S(1073), FSV-T(1073), and FSV-G(1073) DNAs were determined by the dideoxynucleotide chain termination method. The codons for amino acid 1073 are indicated in each case. Y - 1 0 7 3 T - 1 0 7 3 S - 1 0 7 3 G 1 0 7 3 - 140 -5.2.2 Transforming Activity of Wild-type, Mutants and Their Revertant  FSV DNAs. The wild-type (wtFSV), revertants [FSV-Y(1073)] or mutated [FSV-S(1073), FSV-T(1073) and FSV-G(1073)] DNAs were isolated, purified as described in Chapter 4 and transfected onto rat-2 cells using the calcium phosphate coprecipitation technique (Graham and Van der Eb, 1973; Wigler, et a l . , 1979). Foci of transformed cells appeared approximately eight to ten days post-transfection with both wt and the revertant DNAs, but were not obvious until at least 30 days following transfection with the mutated DNAs. Cell lines cloned from foci induced by the FSV mutants were also not as overtly transformed by morphological criteria as cells expressing wt or revertant DNAs (Figure 5.3). Nonetheless, these cells demonstrated anchorage independent growth in soft agar unlike the normal rat-2 cells (data not shown). FSV-S(1073) transformed cells also induced tumors in syngeneic immuno-competent rats, but did so with a longer latent period than wt or revertant transformed cel ls . Therefore changes in the TAT codon for tyrosine-1073 to codons for serine, threonine or glycine resulted in mutant FSV DNAs that were s t i l l transformation-competent, but which transformed cells with a long latent period and induced a less tumorigenic phenotype compared with wt or revertant DNAs. The mutants were similar in biological activity to the previously isolated FSV-F(1073) mutant described in Chapter 4. - 141 -Figure 5.3: Transformation of rat-2 cells following transfection with FSV DNAs. Phase-contrast photomicrographs of rat-2 cells (40x). Cells were transfected with wtFSV or with FSV genomes encoding substitutions at residue 1073. a, serine, FSV-S(1073); b, threonine, FSV-T(1073); c, glycine, FSV-G(1073); d, untransfected rat-2 cel ls; e, tyrosine, wtFSV. - 142 -- 143 -5.2.3 Expression, Structure and In Vitro Kinase Activities of wt and  Mutant Proteins The transformed cells isolated following transfection with wt or mutant FSV DNAs were examined for P 1 3 0 g a g - f p s expression, phos-phorylation and associated kinase activity. To identify P lSO^g - -^ 8 q c cells were metabolical ly labelled with [ S] methionine and lysates were immunoprecipitated with a monoclonal antibody directed against avian pl9^s. The amount of [3 5S] methionine incor-porated reflects the level of P 1 3 0 g a g - f p s expression which varied among the different clones of wtFSV and mutant-transformed cells (Figure 5.4a). A number of- observations suggested that the long latent period for transformation and distinctive morphology associated with cells transformed by mutant FSV DNAs were due to structural changes introduced into P130 g aS~* p s, rather than decreased expression levels. First , a clone of transformed cells isolated following trans-fection with revertant FSV-Y(1073) DNA had approximately 5-fold less p l 3 0 gag- fps than a clone of mutant transformed cells, yet the latter cells were not as overtly transformed as the revertant trans-formed cells (data not shown). Second, a clone of FSV-G(1073) transformed cells had about 3-fold more P 1 3 0 g a g _ f p s than a clone of FSV-T(1073) transformed cells (Figure 5.4a), yet the FSV-G(1073) transformed cells were flatter and less refractile than the FSV-T(1073) - 144 -Figure 5.4: Expression of wt; and mutant p l 3 0 gag- fps in trans-formed rat-2 cells and quantitation of their respective tyrosine protein kinase activit ies. 4a: Normal or transformed rat-2 cells were labelled for 16 hours with OCT 100 ucl [ S]methionine, immunoprecipitated with a n t i - p l 9 g a g serum and examined by gel electro-phoresis to identify PlSOgag-fps^ Lane A, rat-2 cells; lane B, FSV-T(1073); lane C, FSV-<J(1073); land D, FSV-S(1073); lane E, wtFSV. 4b: Cells from duplicate unlabelled cultures were immunoprecipitated in an ident-ical fashion and then incubated with 5 ug of acid-denatured enolase in the presence of lOmM MnCl2 and 2.5 uci [tf-3 2P]ATP for 15 minutes at 30°C. Samples were analysed by electrophoresis through a 7.5% SDS-polyacrylamide gel and autoradiography. Lane F, rat-2 cel ls; lane G, FSV-T(1073); lane H, FSV-G(1073); lane I, FSV-S(1073); lane J , wtFSV. - 146 -transformed cells (Figure 5.3). In order to determine the relative kinase activities of the wt and mutant FSV PISO63^""^8 proteins, enolase was included in the immune complex kinase reactions as an exogenous substrate for tyro-sine phosphorylation (Figure 5.4b). Quantitation of the extent of enolase phosphorylation by mutant and wt PISO 2 9^ -"^ 8 proteins revealed that the kinase activities of FSV-S(1073), FSV-T(1073) and FSV-G(1073) proteins were 6.8, 4.7 and 6.3 fold lower, respectively, than that measured for wtPlSC^^^P 8 . The tyrosine protein kinase activity of the wild-type and mutant proteins was quantitated by deter-q p mining the ratio of °^P-enolase, labelled by an in vitro kinase reaction to that of the respective PISO 2 3^ -"^ 8 metabolically labelled with [35S]methionine (Figure 5.4). This ratio represents the extent of kinase activity per amount of PISC^9^"""^8 present in the reaction, and the values reported are an average of four indepen-dent experiments. Cell lines that were cloned from each of the revertant FSV-Y(1073) transformed cells were shown to express P 1 3 0 g a g - f p s with similar in vitro kinase activities and electrophoretic mobilities as wtFSV P I S O 6 3 6 - ^ 8 (data not shown). Both biological and biochemical data indicated that the PlSOgas-^PS proteins encoded by the revertant DNAs were structurally and functionally identical to - 147 -wtFSV P130 g a g " " f p s and therefore the revertant FSV-Y(1073) transforming proteins were not examined further. Several lines of evidence show that the serine-1073 of FSV-S(1073) PISO 6 ^"^ 8 and the threonine-1073 of FSV-T(1073) P130 g a g ~^ p s do not become phosphorylated either in the rat-2 cells expressing these proteins or in vitro during immune complex kinase reactions. The electrophoretic mobilities of the mutant proteins were slightly greater than those of the wild-type PISO 6 8 - 8"^ p s , which may result from decreased phosphorylation (Figure 5.4). Autophosphory-lation of the mutant proteins in vitro was reduced compared with that of wtFSV P 1 3 0 g a g _ f p s (Figure 5.4b), consistent with the loss of the major autophosphorylation site. Conclusive evidence that residue 1073 was not phosphorylated in mutant proteins containing serine or threonine at this site was provided by comparing two-dimensional maps qp of °^P-labelled tryptic phosphopeptides from mutant and wild type PiSOgag-fps. Phosphorylated peptides 3a-3c of wtFSV P 1 3 0 g a g - f p s are derived from a single tryptic phosphopeptide con-taining residue 1073 (Chapters 3 and 4) and were clearly absent in tryptic peptide maps of FSV-S(1073) or FSV-T(1073) P 1 3 0 g a g ~ f p s phosphorylated in vitro (Figure 5.5). Similar analysis of P 1 3 0 g a g - f p s immunoprecipitated from cells metabolically labelled qp with °^P-orthophosphate showed that residue 1073 was only phos-- 148 -Figure 5.5: Tryptic phosphopeptide analysis of wt FSV and mutant FSV P 1 3 0 g a g _ f p s proteins labelled in vitro. p1 3 0gag-fps encoded by wild-type and mutant FSVs were labelled in an immune complex kinase assay follow-ing immunoprecipitation with a n t i - p l 9 g a g serum from transformed cel ls . The wt and mutant P 1 3 0 g a g _ f p s proteins were gel purified, digested with trypsin and separated in two-dimensions on thin-layer cellulose plates. Electrophoresis at pH 2.1 was from left to right with the anode on the le f t , and chromatography was from bottom to top. An "0" indicates the sample origin. Tryptic digests were as follows: a, wtFSV; b, FSV-S(1073); c, FSV-G(1073); d, FSV-T(1073). The spot that is not numbered and occurs to the right of 3c is seen only in vitro and probably corresponds to the spot 2 found only in vitro with FSV-L5 P 1 4 0 g a g _ f p s . - 149 -- 150 -phorylated in the wild-type protein (Figure 5.6) consistent with the in vitro results. These tryptic peptide mapping studies support and confirm the assumption that neither serine nor threonine is phosphory-lated when substituted for a tyrosine at residue 1073 within P130Bae - * p s # The retention of residual kinase activity and oncogenic potential by the mutant proteins suggests that they have not suffered gross conformational changes as a consequence of the amino acid sub-stitutions at position 1073. However, the reduced kinase activity and oncogenic potential of these mutant proteins appears to correlate with a lack of phosphorylation at residue 1073 within FSV P 1 3 0 g a g - f p s . 5.2.4 Tyrosine Phosphorylation of wtFSV and Mutant FSV P 1 3 0 g a g ~ f p s  from Transformed Cells Phosphoamino acid analysis of mutant P l 3 0 g a g - f p s pro-op teins isolated from P-labelled cells showed that the mutant proteins contained substantially less phosphotyrosine than wt p l 3 0 gag- fps (Figure 5.7). This is consistent with the tryptic phosphopeptide mapping results of the mutant proteins isolated from qp P-metabolically labelled transformed cells and further confirms that the major site of tyrosine phosphorylation (Figure 5.6; spots 3a and 3c) was destroyed in these mutated P 1 3 0 g a g - f p s proteins. However, the loss of tyrosine-1073 did not appear to qualitatively - 151 -Figure 5.6: Tryptic phosphopeptide analysis of P 1 3 0 g a g ~ f p s encoded by wild-type and mutant FSVs. FSV p l 3 0 gag- fps ^ 5 labelled with 3 2 P in vivo by incubation of wtFSV transformed cells with 32 P-orthophosphate for 12 hours and mutant FSV o o transformed cells with P-orthophosphate for 4 hours. The 3 2 P-labelled P 1 3 0 g a g _ f P s proteins were isolated by subsequent immunoprecipitation of the labelled protein with a n t i - p l 9 g a g serum. Gel-purified PISO^^^P8 1 was then digested with trypsin and separated by electrophoresis at pH 2.1 in the f i rs t dimension and chromatography in the second dimension. The anode is to the left and the cathode to the right. Tryptic digest were as follows: A, wtFSV; B, FSV-S(1073); C, FSV-G(1073); and D, FSV-T(1073). - 152 -- 153 -Figure 5.7: Phosphoamino acid analysis of wtFSV and mutant FSV P 1 3 0 g a g _ f p s proteins. Rat-2 cells transformed with wtFSV or mutant FSV [FSV-S(1073); FSV-T(1073); FSV-G (1073)] were labelled with 32P± for 12 hours, lysed, immunoprecipitated with an t i - p l9 g a g serum and the immunoprecipitates separated by gel electro-phoresis. 3 2 P-labelled P 1 3 0 g a g - f p s was recov-ered from the gels, acid-hydrolyzed and analysed for phosphoamino acids by two-dimensional electrophoresis at pH 1.9 and . pH 3.5 followed by autoradiography. The positions of marker phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) as revealed by ninhydrin staining are indicated. A, wtFSV; B, FSV-S(1073); C, FSV-G(1073); and D, FSV-T(1073). The number of Cerenkov counts loaded were as follows: wtFSV, 1300 cpm; FSV-S(1073), 1100 cpm; FSV-G(1073), 540 cpm; and FSV-T(1073), 308 cpm. - 154 -- 155 -affect the phosphorylation of other tyrosine and serine residues within PiSQgag-fps^ The mutant Piao 6 8 8 -*** 8 proteins were s t i l l phosphorylated at tryptic phosphopeptide 4, which represents the other major tyrosine site in wtFSV P 1 3 0 g a g - f p s phosphorylated only in vivo. Also, the minor and variable gag tyrosine site (peptide 1) was phosphorylated in the mutant proteins, although this phosphopeptide was not obvious in the map of FSV-T(1073) P 1 3 0 g a g ~ f p s shown in Figure 5.6. In addition, the uncharacterized phosphotyrosine contain-ing peptide 6 and the major phoshoserine sites (peptides 5 and 8) were demonstrably phosphorylated in the mutant proteins. In fact, in a series of independent tryptic digests, metabolically labelled for 4 •Dp hours or 12 hours, with P i f the only consistent difference between the tryptic phosphopeptides of wtFSV and mutated PISO 6 3 ^ - ^ 8 proteins was the absence of 3a-3c from the mutant proteins [FSV-S(1073); FSV-T(1073); and FSV-G(1073)]. These tryptic phosphopeptide maps are similar to those reported for the previously isolated FSV-F(1073) mutant P 1 3 0 g a g - f p s described in the preceding chapter. Taken together these data suggest that the gross structure of these mutant proteins was probably maintained in the presence of the changes at position 1073 within P I S O ^ " ^ 8 5.3 Discussion The fact that neither serine nor threonine were phosphory-lated when placed at a tyrosine kinase recognition site is a satisfying - 156 -demonstration of the specificity of the p l 3 0 gag- fps protein kinase activity for tyrosine residues. While i t seems probable that the amino acids surrounding tyrosine-1073 of wt P130gag-fps and other tyro-sines are important in targeting the kinase to a particular residue (Hunter, 1982; Pike, et a l . , 1982; Wong and Goldberg, 1983b; Braun, et a l , 1984), i t is apparent that there is a str ict requirement for tyrosine at the site of phosphorylation. It is interesting to speculate as to the reasons for this specificity. The mechanisms for transferring a phosphate group to the phenolic hydroxyl of a tyrosine residue may be quite different from those required to phosphorylate the less reactive aliphatic hydroxyIs of serine or threonine. Although as discussed previously in Chapter 1, there is an obvious sequence relationship between the enzymatic domains of tyrosine protein kinases such as FSV p l 3 0 gag- fps and those kinases specific for serine and threonine residues, such as the cata-lyt ic subunit of cAMP-dependent protein kinase. This suggests that the basic biochemical events involved in phosphorylation may be similar for a l l the hydroxyamino acids. In fact, kinetic studies suggest similar mechanisms of phosphate transfer whether the nucleophilic hydroxyl is on a serine or tyrosine residue (Erneux, et a l . , 1983; Bolen, et a l . , 1980; Wong and Goldberg, 1984). An alternative explanation might be that the active site of p l 3 0 gag- fps may be folded such that only a tyrosine at residue 1073 can be positioned to accept a phosphate group. Sub titutions at position 1073 might also disru t a conformati nal signal formed by the surrou ding recog ition sequ nce. - 157 -Amino acid substitutions at the conserved tyrosine-1073 residue of P 1 3 0 g a g ~ f p s did not completely abolish i ts activity. I have changed tyrosine-1073 to serine, threonine, glycine and phenyla-lanine residues and have found that a l l of these mutant FSV genomes induced cells of nearly identical transformed phenotype albeit after a long latent period. In addition, these transformed cells expressed PlSO^g - -^ 8 with a similar reduced biochemical activity. These data indicate that phosphorylation of this highly conserved tyrosine does not have an essential function in catalysis, but rather may play a regulatory role. The similar effects of these mutations was surprising con-sidering that the R-groups of the substituting amino acids are quite different; phenylalanine possesses a hydrophobic aromatic ring, serine and threonine have polar aliphatic side chains, while glycine lacks a side chain and can introduce greater f lex ib i l i ty into the polypeptide. These considerations imply that the important characteristic of tyrosine-1073 is i ts abil ity to become phosphorylated. I have there-fore concluded that the reduced kinase activity and transforming potential of the mutant proteins are not due to disruptive changes in protein conformation, but are a consequence of the absence of phos-phorylation. Although tyrosine-1073 appears to be important for the regulation of pisogag-^P 8 tyrosine kinase activity, there are - 158 -additional tyrosine, serine and threonine phosphorylation sites within the PISO 8 3 - 6"^ 8 of FSV transformed cells which may also have a regulatory function. Therefore, the activity and function of the FSV transforming protein may be subject to modulation by a number of cellular kinases, each with different amino acid substrate specifities. - 159 -CHAPTER 6 6.0 Site-directed Mutagenesis of Lysine-950 Within PlSQgag"^8  Eliminates Both Its Kinase Activity and Transforming Abil ity. 6.1 Introduction To understand more fully the biological roles of tyrosine protein kinases, i t is important to identify the specific structural domains or amino acid residues that contribute to the enzymatic activities of these proteins. The catalytic domains of p 6 0 s r c , P140 g a g~"^ p s and the EGF receptor have been partially defined by limited tryptic digestion (Levinson, et a l . , 1981; Weinmaster et a l . , 1983; Brugge and Darrow, 1984; Basu et a l . , 1984). Since these cleavage fragments possess tyrosine kinase activity, they must, by definition, contain the active center which functions in ATP binding and phosphotransfer. Barker and Dayhoff (1982) have shown that the cyclic-AMP dependent serine protein kinase (cAPK) and the p 6 0 s r c tyrosine protein kinase are distantly related, based on sequence homology within their respective catalytic domains. More specifically, this homology includes a lysine residue that is proposed to function in ATP-binding. - 160 -The identity and putative function of this lysine was determined by studies using the reactive ATP analogue, p-fluoro-sulfonylbenzoyl 5' - adenosine (FSBA). Affinity labels such as FSBA are compounds that bind specifically and reversibly to a protein due to their complementarity with the active site and then react covalently with one or more amino acid residues within the active site by virtue of a small reactive group (Singer, 1977). The structure of FSBA is similar to that of ATP, except that the triphosphates have been replaced by a side chain of comparable size that contains a reactive sulfonylfluoride group in the position of the X-phosphate (Figure 6.1). FSBA can react with the free electron pair of lysine, tyrosine, histidine (Zoller, et a l . , 1981) and cysteine (Togaski and Reisler, 1982), yielding an adduct of the sulfonylbenzoyladenosine moiety of FSBA and the amino acid. The use of affinity labels such as FSBA provides a means for locating specific regions of the protein that are potentially involved in binding or catalysis. FSBA was shown to bind specifically to lysine-71 within cAPK (Zoller, et a l . , 1981) and lysine-295 within p 6 0 s r c (Kamps, et a l . , 1984). Covalent modification of these kinases with FSBA results in enzymatically inactive proteins. FSBA also inactivates the serine-specific cGMP-dependent protein kinase (cGPK), by covalently modifying the equivalent lysine in i ts ATP binding site (Hashimoto, e t a l . , 1982). FSBA has also been useful in studies concerning the - 161 -Figure 6.1: Comparison of the structure of p-fluorosulfonylbenzoyl-5'-Adenosine (FSBA) and Adenosine 5'-triphosphate (ATP). CHo Adenine .0 OH OH FSBA C H 9 Adenine .0. ATP OH OH - 163 -nucleotide binding sites of casein kinase II (Hathaway, et a l . , 1981) and the EGF receptor (Buhrow, et a l . , 1982; Buhrow, et a l . , 1983; Basu, et a l . , 1984). Therefore, i t appears that the tertiary structures of the ATP binding regions within the cAMP-dependent and cGMP-dependent serine kinases, as well as the p 6 0 s r c tyrosine protein kinase a l l orient a homologous lysine residue such that i t reacts with FSBA. These f ind-ing provide strong evidence that the sequence homology found between p 6 0 s r c and the catalytic subunit of cAPK, reflect structural as well as functional homology and support the idea that protein kinases, irrespective of their amino acid substrate specificity, share a common ancestry. Similarly, the C-terminal kinase domain of P 1 3 0 g a g - f p s contains a lysine at position 950 which is homologous to the highly conserved lysine-295 of p 6 0 s r c (Shibuya and Hanafusa, 1982), which has been proposed to function as an ATP binding site in p 6 0 s r c (Kamps, et a l . , 1984). To assay for the presence of amino acid residues which participate in ATP binding, I have used FSBA to covalently modify the ATP binding site within P l 4 0 g a g ~ " f p s . In addition, to investigate more specifically the requirement for lysine-950 within P 1 3 0 g a g _ f p s , I have used the technique of - 164 -oligonucleotide-directed mutagenesis. In collaboration with M. Zoller, lysine-950 within P 1 3 0 g a g _ f p s has been mutated in order to evaluate the contribution of this residue in the phosphotransfer reaction. If the kinase activity intrinsic to P 1 3 0 g a g - ^ p s i s crucial for i t s transforming function, then alterations in i ts ATP binding site should eliminate not only i t s kinase activity, but also i t s transforming potential. 6.2 Results 6.2.1 Inactivation of P 1 4 0 g a g " f P s Kinase Activity by Treatment  with FSBA In order to measure the inactivation of P l 4 0 g a g ~ ^ p s kinase activity by FSBA over time, immune-precipitates prepared from FSV-L5 transformed CEF were incubated at 37°C with 1.7 mM FSBA. A l i -quotes were removed at timed intervals following the addition of FSBA, placed on ice, incubated in the presence of f£-32P]ATP as described in section 2.4 and the P-labelled proteins resolved by SDS-PAGE (Figure 6.2A, lanes 1-6). To control for nonspecific loss of P 1 4 0 g a g - f p s kinase activity during incubation at 37 °C, P140 g a g ~^ p s immune-precipitates were not treated with FSBA, but were incubated at 37°C, sampled at the start and finish of the experi-ment and assayed as for the FSBA treated samples (Figure 6.2A; lanes 7 - 165 -Figure 6.2 Inactivation of p i 4 0 g a g ~ f p s kinase activity by treatment with FBSA A. FSBA inactivation of P140gag-fps kinase activity over time: Immunopre-cipitates from FSV-L5 transformed cells were incubated at 37°C in the presence or absence of 1.7 ml FSBA, sampled at various times, placed on ice, kinased and the 32 P-labelled proteins resolved by electrophoresis through a 15% SDS-polyacrylamide gel. Lanes 1 through 6 represent incubation times of 0, 10, 30, 45, 60 and 90 minutes respectively. Lane 7, immunoprecipitates left on ice for 90 minutes in the absence of FSBA; lane 8, immunoprecipitates incubated at 37 °C for 90 minutes in the absence of FSBA; lane 9, purified catalytic subunit of cAPK (CAT) incubated at 37°C for 90 minutes in the absence of FSBA and kinased in the presence of histones; lane 10, CAT incubated in the presence of 1.7 mM FSBA for 90 minutes at 37 °C and kinased in the presence of histones. B. Dose response of p i 4 0 g a g ~ ^ p s kinase activity for FSBA inactivation: Immunoprecipitates from FSV-L5 transformed cells were incubated in the presence of various concentrations of FSBA at 37 °C for 60 min-utes, kinased, and analysed by 7.5% SDS-PAGE followed by autoradiography. Lanes 1, immunoprecipitates untreated with FSBA and incubated on ice for 60 minutes. Lanes 2 through 8 represent immunoprecipitates treated with final concentrations of 0, .1 , .2, .5 , 1.0, 1.5, and 2.0 mM. FSBA respectively. - 166 -- 167 -and 8). To test whether the synthesized FSBA used in these experiments was chemically reactive, purified type II catalytic subunit from porcine heart muscle (CAT) (obtained from M. Zoller) was treated with FSBA and i ts residual phosphotransferase activity was measured by the addition of histone type II (Sigma) to the kinase assay and compared with the kinase activity intrinsic to untreated CAT (Figure 6.2A; lanes 9 and 10). To quantitate the extent of enzyme inactivation resulting from treatment of P i 4 0 g a g ~ f P s with FSBA, 3 2P-labelled pro-teins were excised from the gel and the amount of 32P-incorporated was determined by Cerenkov counting. Pre-incubation of p i 4 0 g a g - f p s with FSBA for 90 minutes at 37°C results in only 8% residual kinase activity, while 35% residual activity was detected with pi40 g a S~ f P s that had not been treated with FSBA. This represents a 4.3-fold decrease in P i 4 0 g a g - f P s kinase activity that can probably be attributed to FSBA inactivation. The inactivation of CAT by FSBA was not quantitated since there was an obvious and significant decrease in o o incorporation of into histones following treatment of CAT with FSBA. Therefore, pre-incubation of p i 4 0 g a g " f p s with 1.7 mM FSBA decreased the phosphotransferase activity in a time-dependent fashion. To demonstrate that the loss in P i 4 0 g a g - ^ P S kinase a c t i -vity was actually due to inactivation of the enzyme by FSBA, the dose response for FSBA inactivation of P 1 4 0 g a g - f P s kinase activity was - 168 -determined. Immunoprecipitates were prepared as described above and incubated at 37 °C for 60 minutes in the presence of various FSBA con-centrations (Figure 6.2B; lanes 2 through 8). Lane 1 represents an untreated P l 4 0 g a g ~ f p s containing immune-precipitate that was left on ice during the incubation period. The results indicate that an increase in FSBA concentration can be correlated with a decrease in kinase activity, which suggests that FSBA inactivates P 1 4 0 g a g - : f p s by reacting with a portion of the protein that functions in ATP bind-ing. Although the inactivation may be due to a nonspecific modifica-tion of the protein by FSBA i t i s important to note that the kinase activity of P 1 4 0 g a g ~ f p s i s stable in the presence of phenylmethyl-sulfonylfluoride (PMSF) (data not shown), a nonspecifically reactive compound that contains the same phenylsulfonylfluoride moiety as FSBA. The failure of PMSF to destroy kinase activity suggests that inactiva-tion due to random reactions of the enzyme with the sulfonylfluoride group of FSBA probably does not occur. These preliminary experiments provided strong indirect evidence that P140g a g~-^PS contains an ATP binding site that may be modified by FSBA. However, to conclusively prove that FSBA reacts with the active site of p i 4 0 g a g ~ f p s one would have to demonstrate that the inactivation could be specifically inhibited by ATP. - 169 -6.2.2 Site-directed Mutagenesis of Lysine-950 Within the Putative  ATP-binding Site of pispgag-fps I have expanded the mutagenesis studies of FSV to include the lysine (K) at position 950 within P 1 3 0 g a g - f p s , proposed to func-tion in ATP binding. Lysine-950 within PISO 6 9 - 6 "^ 8 was changed to arginine (R) or glycine (G) using oligonucleotide-directed mutagenesis of the M13mplO-FSV clone (in collaboration with M. Zoller). These mutations were chosen for the following reasons. Since the positive charge carried by the £-amino group of lysine-950 may be important in ATP binding and/or catalysis, the amino acid arginine was chosen as an analogous substitute, since i ts side chain is also positively charged. However, i f the size and shape of lysine-950 is absolutely cr i t ica l to produce the required tertiary structure for a functional active site, then the substitution of arginine for lysine may result in an enzymatically inactive tyrosine protein kinase. On the other hand, the glycine mutation represents a control for ATP binding at residue 950. Since i ts substitution for a lysine residue results in an amino acid that lacks an R-group which can participate in ATP binding, i t should yield a protein deficient in phosphotransferase activity. The oligonucleotides designed to direct the desired changes within the FSV genome are shown in Figure 6.3. Mutagenic oligo-- 170 -Figure 6.3: The synthetic oligonucleotides used to mutate the codon for lysine-950 of FSV P 1 3 0 g a g - f P s . Oligonucleo-tides 17 long were designed to alter the codon for residue 950 by one or two nucleotides to code for an arginine or a glycine as indicated. The mutagenic oligonucleotide encoding the wild-type codon AAA was used to revert FSV-R(950) to FSV-K(950). The figure displays nucleotides 3212-3236 of the wt FSV nucleotide sequence (Shibuya and Hanafusa, 1982). - 171 -ACOXCGTCKXXXJTGAMTOCTCC . . . 3' 5 * HXGTGGCGGTGAGATCC-3' 51 -CCGTGGCGGTGGGATCC-3 * 5' -CXXTGGCGGTGAMTCC-3 * LYS Wild-type FSV ARG FSV-R (950) GLY FSV-G (950) LYS FSV-K (950) - 172 -nucleotides 17 nucleotides long were synthesized to alter the AAA codon for lysine-950 to an AGA codon for arginine and a GGA codon for gly-cine. Apart from the single base substitution of a G for A in the case of FSV-R(950), and the two base change of GG for AA in the case of FSV-G(950), the synthetic mutagenic primers are identical with nucleo-tides 3216-3233 of the FSV genome as defined by Shibuya and Hanafusa (1982). The M13mplO-FSV clone, mutagenesis procedure, screening and propagation of mutants was exactly as described in the preceding two chapters. Once again to control for second-site mutations that could have occurred during the mutagenesis procedures and/or the propagation of mutant phage, the FSV-R(950) genome was reverted using an oligo-nucleotide which encoded the wild-type sequence (Figure 6.3). 6.2.3 Transfection of Rat-2 Cells with Wild-type, Mutant or Revertant  FSV DNAs To test whether the FSV mutated genomes possessed transform-ing activity and also to ensure that the revertant FSV-K(950) DNA had the same transforming potential as the wild-type FSV DNA, rat-2 cells were transfected with the wtFSV, FSV-R(950), FSV-G(950), or FSV-K(950) DNAs. These transfected cells were cultured in DMEM supplemented with 5% calf serum and 0.5 uM dexamethasone, and were examined daily for the appearance of foci . Approximately 10 days following transfection of rat-2 cells with wtFSV DNA or revertant FSV-K (950) DNA, foci contain-ing transformed cells with a round refractile morphology were obvious - 173 -on a background of normal f lat rat-2 cel ls . However, at 10 weeks post-transfection, rat-2 cells transfected with mutant FSV-G(950) or FSV-R(950) DNAs did not appear to be different from control cultures transfected with rat-2 carrier DNA only, which suggested that these mutated FSV DNAs were nontransforming. The revertant FSV-K(950) DNA had the same transforming potential as the wtFSV DNA, which indicated that alterations in the codon for lysine-950 eliminated the transforming activity of FSV DNA. Foci of transformed rat-2 cells that appeared after transfection with wtFSV or revertant FSV-K(950) DNAs were picked, cloned in soft agar, and expanded in mass culture. The morphological phenotype of the revertant transformed cel l lines was indistinguishable from that of the wild-type FSV cel l line (Figure 6.4; panel B and D), and both of these FSV transformed cel l lines formed large colonies in soft agar (data not shown). As expected, the cells cloned from these two FSV transformed cel l lines were very round and refractile, unlike the normal rat-2 cells which were characteristically very f lat and somewhat transparent (Figure 6.4; panel A and panel C). Since foci were selected for transformed phenotype and since the changes at residue 950 had apparently eliminated the transforming activity of p1 3 0gag-fps I cotransfected the mutated FSV genomes with an alternative selectable marker. Rat-2 thymidine kinase (TK) minus cells were cotransfected with the Herpes simplex type 1 thymidine - 174 -Figure 6.4: Morphological phenotypes of rat-2 cells following trans-fection with FSV DNAs. Phase-contrast photomicrographs of rat-2 cells (40x). Cells were transfected with wt FSV, revertant FSV-K(950) FSV genomes or cotransfected with mutant FSV-R(950) DNA plus pTK .^ Following selection in HAT-DMEM containing 10% FBS or DMEM plus 5% CS and .5uM dexamethasone cells were cloned and grown in mass culture. Panel A, normal rat-2 cells; panel B, wt FSV transformed cells; panel C, FSV-R(950) transfected rat-2 cel ls; and panel D, revertant FSV-K (950) transformed cel ls . - 176 -kinase gene (pTK-^ ) together with wild-type FSV, mutant [FSV-R(950) or FSV-G(950)] or revertant [FSV-K(950)] 4.7 kb inserts as described in section 2.9 and placed under HAT selection 48 hours post-transfection. The resulting HAT selected colonies were cloned from the original plates and grown in 35 ran wells for further analysis. The HAT resistant cells transf ected with the mutant DNAs were similar in appearance to rat-2 ce l ls , however, approximatley 50% of the HAT selected clones from cells transfected with wt FSV or revertant FSV DNAs had a transformed phenotype. 6.2.4 Expression and Characterization of Mutant FSV-R(950) and  FSV-G(950) P 1 3 0 g a g " f p s Proteins Fifteen HAT resistant clones originating from rat-2 cells cotransfected with pTK^  and FSV-R(950) or FSV-G(950) DNAs were selected and tested for expression of P 1 3 0 g a g - f p s . Equal numbers of cells were labelled with [3^S]methionine for 16 hours, lysed, immunoprecipitated with a n t i - p l 9 g a g serum and examined for the presence of P 1 3 0 g a g - f p s by SDS-PAGE and fluorography. Of the 15 different clones tested from each set of FSV-R(950) or FSV-G(950) transfected cel ls , only one clone from each set was found to express piSOgag-fP8 (Figure 6.5; panel A). The figure also shows two TK positive rat-2 lines [R-2(tk)] which are negative for P 1 3 0 g a g _ f p s expression and a wt FSV P 1 3 0 g a g " f p s expressing cel l line (K-950). The cells expressing FSV-R(950) and FSV-G(950) P I S O ^ " ^ 8 pro-- 177 -Figure 6.5: Analysis of wild-type and mutant FSV transfected rat-2 cells for FSV PiaO88,8""*138 synthesis and tyrosine protein kinase activity. R-950 and R-2 (tk) indicate rat-2 cells that were transfected with pTK^ P l u s FSV-R (950) DNA and HAT selected. G-950 and R-2 (tk) indicate rat-2 cells that were cotransfected with pTK^  plus FSV-G(950) DNA and selected in HAT-DMEM, K-950 indicates wt FSV transformed rat-2 cel ls . R-2 indicates normal rat-2 cel ls . A. Rat-2 cells cotransfected with pTK-^  plus mutant FSV-R(950) or FSV-G(950) DNAs were selected in HAT-DMEM. HAT resistant clones were grown in 35 mm wells, labelled for 16 hours with 100 uCi [ 3 5 S]-methionine, immunoprecipitated with a n t i - p l 9 g a g serum and examined by gel electrophoresis and fluoro-graphy to detect PlSOgag-fps. B. Normal rat-2 cells and FSV-R(950) cells were labelled for 16 hours •30 with 1 mCi of P-orthophosphate, processed as described in section 2.3 and analysed by SDS-PAGE (7.5%). C. Cells from unlabelled cultures were immuno-precipitated as described above and then incubated with 5 ug of acid-denatured enolase in the presence of 10 mM MnCl2 and 2.5 uCi [ $-3 2P]ATP for 15 minutes at 30°C. Samples were analysed by electrophoresis through a 7.5% SDS-polyacrylamide gel and autoradiography. - 179 -teins had a normal morphological phenotype similar to that of rat-2 cells (Figure 6.4; panels A and C). Consistent with this f lat normal morphological phenotype, neither FSV-R(950) nor FSV-G(950) rat-2 cells displayed anchorage independent growth, as judged by their inability to grow in soft agar (data not shown). To determine whether these mutant p l 3 0 gag- fps proteins had kinase activity, wild type FSV and mutant [FSV-R(950) or FSV-G (950)] P 1 3 0 g a g - f p s proteins were immunoprecipitated from equal numbers of unlabelled cells and the immune complexes were incubated with MnCl2, [)(-32P]ATP, and soluble denatured rabbit muscle enolase (Figure 6.5; panel C). In this reaction wtFSV p l 3 0 gag- fps (K-950) is autophosphorylated and also phosphorylates enolase • at a single tyrosine residue (Cooper, et a l . , 1984a). However, neither of the mutant cel l lines (R-950 nor G—950) demonstrated detectable autophosphorylation of P 1 3 0 g a g - f p s or phosphorylation of the exogenous substrate enolase; the background from both of these reactions is similar to that found with normal rat-2 cells (Figure 6.5; panel C). This experiment indicates that alterations in the codon for lysine-950 within p l 3 0 gag- fps yields an enzymatically inactive tyrosine protein kinase. In FSV-transformed cel ls , P130gag-fps is phosphorylated mainly at serine and tyrosine residues, and to a lesser degree at threonine residues (see Figure 5.7; panel A). In order to determine - 180 -whether mutant FSV-R(950) P 1 3 0 g a g - f p s was phosphorylated in cells expressing this protein, both normal rat-2 cells (R-2) and mutant FSV-R (950) rat-2 cells (R-950) were metabolically labelled with 3 2 P -orthophosphate for 12 hours, lysed, immunoprecipitated with ant i -piggag serum, and the labelled proteins resolved by SDS-PAGE. The data show that mutant R-950 P 1 3 0 g a g ~ : f p s was found to be phosphory-lated in intact cells (Figure 6.5; panel B). Since the mutant protein is both stably expressed and phosphorylated these data suggest that the substitution of arginine for lysine-950 does not substantially disrupt the conformation of the protein. To investigate the phosphorylation sites pf the mutant pro-tein, purified in vivo 3 2P - label led FSV-R(950) P 1 3 0 g a g ~ f p s was subjected to tryptic peptide analysis. Inspection of the FSV-R-(950) P 1 3 0 g a g ~ ^ p s tryptic map indicates that i t is missing the major site of tyrosine phosphorylation, tyrosine-1073 (Figure 6.6; panel C). This site is contained within tryptic phosphopeptides 3b and 3c of wtFSV P l S C r 5 8 6 ^ ^ (Figure 6.6, panel A). In fact, the FSV-R (950) P 1 3 0 g a g - f p s tryptic map is similar to the tryptic phospho-peptide map of the mutant FSV-S(1073) P 1 3 0 g a g _ f p s (Figure 6.6; panel B) in which the major tyrosine phosphoaceptor site (tyrosine-1073) has been destroyed (Chapter 5 ) . Interestingly, FSV-R (950) P 1 3 0 g a g ~ f p s appears to be phosphorylated at tryptic phosphopeptide 4, which represents the other major tyrosine site which is phosphory-lated exclusively in vivo in both wt FSV P 1 3 0 g a g - f p s and in a l l - 181 -Figure 6.6: Comparison of tryptic phosphopeptides from p l 3 0 gag- fps encoded by wild-type FSV, mutant FSV-S(1073) and mutant FSV-R (950). Cells were labelled op with 1 mCi P-orthophosphate for 12 hours, lysed, immunoprecipitated and p l 3 0 gag- fps was identified as described in the legend for Figure 6.5. Gel-purified PISO^^-^P8 was then digested with trypsin and separated by electrophoresis at pH2.1 on thin-layer cellulose plates in the f i rs t dimension and chromato-graphy in the second dimension. Tryptic digests were as follows: A, wt FSV; B, FSV-S(1073); and C, FSV-R(950). - 182 < - 183 -the FSV PISO 8 9 ^ - ^ 8 proteins mutated at tyrosine-1073: FSV-S(1073) (Figure 6.6; panel B), FSV-G(1073) (Figure 5.6, panel C) and FSV-T (1073) (Figure 5.6; panel D). L i t t le is known about the other sites of phosphorylation: spots 5, 6, 7, 8 and 9 (Figure 6.6). Except for spots 5 and 8, the other spots represent variable and minor sites of phos-phorylation. However, spot 5, and probably spot 8, contain phospho— serine, and spot 6 is known to be a minor site of tyrosine phosphoryla-tion (Chapter 3). Presumably the majority of these uncharacterized sites of phosphorylation contain phosphoserine, and possibly some phospho-threonine, since phosphoamino acid analysis of FSV-R(950) P 1 3 0 g a g - f p s yields mainly phosphoserine, minor amounts of phospho-threonine and trace levels of phosphotyrosine (data not shown). How-ever, the use of vanadate as an inhibitor of phosphotyrosine specific phosphatases during in vivo labelling of FSV-R(950) rat-2 cells increases the detection of phosphotyrosine comparable to that of phos-phothreonine, confirming that this mutant protein is phosphorylated at tyrosine (I. MacLaren, personal communication). Except for the phosphorylation of tryptic peptide 1, which is a variable site of autophosphorylation, the tryptic phosphopeptides from in vivo 3 2P-labelled FSV-S(1073) P 1 3 0 g a g _ f p s and FSV-R (950) P 1 3 0 g a g _ f p s have similar mobilities (Figure 6.6). This indicates that FSV-R(950) P ISO 6 ^"^ 8 is only missing the sites of autophosphorylation consistent with i ts lack of kinase activity when - 184 -assayed in the immiine complex reaction (Figure 6.5, panel C). The fact that FSV- R(950) P l 3 0 g a g - f p s contained sites of phosphorylation related to both wtFSV p i 3 0 g a g - f p s and mutant FSV-S(1073) p l 3 0 gag- fps argues that the mutation of residue 950 does not have a global effect on the protein's native structure. However, the lack of detectable kinase activity exhibited by the mutant FSV-R(950) pro-tein suggests that changes at position 950 within P 1 3 0 g a g - f p s affect the protein's abil i ty to function in the phosphotransfer reaction. 6.2.5 Trans-Phosphorylation of FSV-R(950) P 1 3 0 g a g ~ f p s by  FSV P 1 4 0 g a g - f p s Since tyrosine-1073 is the major site of autophosphorylation i t is not surprising that this site is not phosphorylated in an enzyma-t ical ly deficient P 1 3 0 g a g - ^ p s protein. However, to ensure that the lack of autophosphorylation within this mutant protein was actually due to an inactive kinase rather than a change in protein structure which prevented phosphorylation at this site, I determined whether FSV-R(950) P 1 3 0 g a g - f p s could be phosphorylated in trans by the enzymatically active FSV P 1 4 0 g a g - f p s . Cleared cel l lysates from FSV-L5 transformed rat-1 cells and FSV-R(950) rat-2 cells were pre-pared. One-half of each lysate was combined and the mixture was immun-oprecipitated, while the remainder of each lysate was immunopreci-pitated separately. The precipitated immune complexes were kinased in the presence of MnCl2 and [ -3 2P]ATP at 20°C for 15 minutes, - 185 -following which the P-labelled proteins were separated by SDS-PAGE and visualized by autoradiography. For comparative controls, in vitro kinase reactions of wtFSV P 1 3 0 gag-fps and mutant FSV-S(1073) P 1 3 0 g a g - f P s were also separated on the same gel, while a kinased rat-2 immune-precipitate was included as a background control (Figure 6.7). When FSV p i 4 0 g a g - f P s and FSV-R(950) p i 3 0 g a g _ f P s were coimmunoprecipitated and incubated in the presence of [)(-32P]ATP both proteins were labelled with 3 2 P (Figure 6.7; lane 1). However FSV-R(950) P I S O ^ ^ P 8 was not labelled in the same ATP:phosphotransferase reaction when immunoprecipitated and kinased alone (Figure 6.7; lane 3), while FSV p i 4 0 g a g ~ f p s was labelled under the same conditions (Figure 6.7; lane 2). These data q p indicate that the incorporation of P into FSV-R(950) P130 g a g~^P s i s a result of trans-phorphorylation by the enzymatic activity intrinsic to FSV p i 4 0 g a g " f P s . In order to identify the sites within FSV-R(950) p l 3 0 gag- fps that were phosphorylated in trans by FSV P 1 4 0 gag-fps t h e 3 2 p-iabelled protein was excised, eluted, concentrated and subjected to tryptic peptide analysis. The major tryptic phosphopeptides found in FSV-R(950) P 1 3 0 gag-fps phos-phorylated in trans were those which correspond to the major autophos-phorylation site of wtFSV p i 3 0 g a g ~ f P s (Figure 6.8; 3a and 3b), which is known to contain tyrosine-1073 (Chapter 3 and 4). These data - 186 -Figure 6.7: In vitro phosphorylation of FSV-R(950) p i 3 0 g a g - f P s by FSV P l 4 0 g a g _ f P s . Cell lysates from unlabelled cells were either immunoprecipitated together or separ-ately as indicated below, kinased in the presence of 10 mM MnCl2 plus 5 uCi [^-3 2P]ATP and analysed by electrophoresis through a 7.5% polyacrylamide gel f o l -lowed by autoradiography. The kinase reactions were from the following immunoprecipitated cel l lysates: Lane 1, FSV-L5 (P140 g a g ~ f p s ) transformed cells plus FSV-R(950) rat-2 cells; lane 2, FSV-L5 ( P l 4 0 g a g _ f P s ) transformed cel ls; lane 3, FSV-R(950) rat-2 cel ls; lane 4, wt FSV (PISO^^P 5 3) trans-formed cells; lane 5, FSV-S(1073) transformed cells; lane 6, normal rat-2 cel ls. - 187 -1 2 34 5 6 P140 P130 P130 - 188 -Figure 6.8: Tryptic phosphopeptide analysis of in vitro phosphor-ylated wt FSV pi30^^~fPs and in vitro trans-phosphorylated FSV-R(950) P130 g a g ~ f P s . P140 g ag~ fP s encoded by FSV-L5 and mutant FSV-R (950) P 1 3 0 g a g _ f P s were coimmunoprecipitated and labelled in an immune complex kinase assay following immunoprecipitation with anti-p 1 9gag serum from FSV-L5 transformed cells and FSV-R(950) rat-2 cel ls , wt FSV P 1 3 0 g a g ~ f p s from wt FSV transformed cells was immunoprecipitated and kinased in an identical fashion. 32 The P-labelled gel purified proteins were digested with trypsin and separated in two dimensions on thin-layer cellulose plates. Electrophoresis at pH 2.1 was from left to right with the anode on the left , and chromatography was from bottom to top. Tryptic digests were as follows: A, wt FSV P130 g a g ~ f P s ; B, FSV-R (950) p i 3 0 g a g _ f P s trans-phosphorylated by FSV Pl^gag-fps. - 190 -suggest that the mutant FSV-R(950) P 1 3 0 g a g _ f p s has maintained a conformation around the major tyrosine phosphoacceptor site which per-mits phosphorylation in trans by FSV P 1 4 0 g a g - f p s , and indicates that the phosphotransferase reaction of the FSV transforming protein can be an intermolecular event as previously described (Mathey-Prevot, 1984). 6.3 Discussion The ATP analogue, FSBA, inactivated the tyrosine specific protein kinase activity intrinsic to the FSV transforming protein in a concentration and time dependent manner. FSBA is also known to speci-f ical ly inactive the kinase activities associated with the cycl ic -nucleotide dependent protein kinases cAPK (Zoller, et a l . , 1981) and cGPK (Hashimoto, et a l . , 1982), and the tyrosine specific protein kinase p 6 0 s r c (Kamps, et a l . , 1984). These studies show that inactivation occurs when FSBA reacts with a homologous lysine residue within the putative ATP binding site of these proteins. Therefore, by analogy, one would predict that FSBA also reacts with a residue located in the ATP binding site of the FSV transforming protein. Inspection of the FSV P130 g a g~"^ p s amino acid sequence indicates that lysine-950 is the l ikely target for FSBA binding, since this residue is homologous to the lysine residues known to be covalently modified by FSBA in cAPK, cGPK, and p 6 0 s r c . Although the FSBA inactivation data demon-strates the presence of an ATP binding site within P 1 3 0 g a g ~ f p s , a detailed understanding of this binding site and the spatial relation-- 191 -ships between localized residues such as lysine-950 and ATP binding w i l l only emerge from x-ray crystallography. Although, the tertiary structure of a protein kinase has yet to be determined, the crystal structure of a number of dinucleotide binding proteins is known. These include lactate dehydrogenase (LDH) (Adams, et a l . , 1973) and nucleotide binding proteins such as adenylate kinase (AK) (Pai, et a l . , 1977). A comparison of the amino acid sequence surrounding lysine-950 of p l 3 0 gag- fps with the sequence known to comprise the adenosine binding sites of these enzymes reveals the homologous sequence: Gly X Gly XX Gly, sixteen residues on the N-terminal side of lysine-950 (Rossmann, et a l , 1974; Pai, et a l . , 1977). X-ray crystallographic studies show that this sequence of glycines is found between two B-pleated sheets that form the adenine dinucleotide binding pocket of lactate dehydrogenase (Adams, et a l . , 1971; Rossmann, et a l . , 1974). Apparently the Gly X Gly XX Gly forms a loop which is involved in the proper positioning and binding of the pyrophosphate moiety of NAD. A topological comparison of adenylate kinase with several dehydrogenases suggests that the substrate-binding sites in AK are equivalent to the NAD-binding sites in the dehydro-genases, with ATP corresponding to the adenosine containing half of NAD (Pai, et a l . , 1977). The cluster of glycines has probably been conserved to provide the f lex ib i l i ty needed to accommodate changes in protein structure induced by NAD or ATP binding. In fact, X-ray analysis indicates that the phosphate-binding loop in both AK (Pai, - 192 -et a l . , 1977) and IDE (Adams, et a l . , 1973) is the epicenter of a large conformational change. Alcohol dehydrogenase, glyceraldehyde 3-phos-phate dehydrogenase and glutathione reductase also bind nucleotides as cofactors and these enzymes a l l contain the Gly X Gly XX Gly in a homo-logous position (Wierenga and Hoi, 1983). It i s interesting to note that this array of glycines is also found in the GTP binding protein of p 2 1 c ~ r a s , and mutations in the second gly have been implicated in the oncogenic activation of normal cellular ras (Wierenga and Hoi, 1983). As discussed in Chapter 1, FSV p l 3 0 gag- fps belongs to a family of tyrosine kinases that are a l l related to p 6 0 s r c by a high degree of sequence homology within the domains which catalyze tyrosine phosphorylation. It i s interesting that the characteristic sequence of glycine residues found in the nucleotide binding proteins discussed above is also present in a l l the oncogenic proteins belonging to the src family of tyrosine kinases (see Figure 1.1). In addition, proteins that do not display tyrosine protein kinase activity also contain this sequence. For example, the serine protein kinase cAPK contains the Gly X Gly XX Gly on the N-terminal side of lysine-71, the lysine covalently modified by FSBA (Zoller, et a l . , 1981). Whether or not the presence of the Gly X Gly XX Gly sequence in these protein kinases is a result of the divergent evolution of these proteins from a primordial adenine nucleotide-binding protein (Schulz and Schirmer, 1974, Eventoff and Rossmann, 1975), or reflects an essential sequence - 193 -of evolutionarily conserved amino acids is not clear. The ubiquity and absolute conservation of the Gly X Gly XX Gly and the neighbouring lysine residue in every protein kinase characterized to date further strengthens the argument that lysine-950 within P 1 3 0 g a g - f p s plays an essential role in catalysis. As more direct evidence, the site-directed mutagenesis of the P 1 3 0 g a g _ f p s lysine-950 destroys the tyrosine protein kinase activity intrinsic to FSV P130 g a g ~^ p s . In addition, mutations of lysine-950 also eliminate the transforming activity of PISO 6 9^ -^^ and support the idea that tyrosine protein kinase activity is crucial for transformation by FSV. It has been proposed that the positive charge carried by the £-amino group of lysine may activate or position the )f-phosphate of ATP, which would consequently activate this phosphate for nucleophilic attack by the OH group of a serine, threonine or tyrosine residue (Kamps, et a l . , 1984). However, other characteristics of this lysine residue must also be important for enzymatic activity since the positively charged arginine residue could not substitute for lysine-950 within P 1 3 0 g a g - f p s . The stable expression of the mutant FSV-R(950) P 1 3 0 g a g - f p s protein, with an arginine substituted for lysine-950 and the abil i ty of this protein to be phosphorylated both in vivo and - 194 -in vitro suggests that this single amino acid change does not grossly affect the structure around the phosphorylation sites within the protein. However, the lack of kinase and transforming activities exhibited by the FSV-R(950) mutant suggests that this conservative amino acid change may perturb the local conformation of the ATP-binding site. It is interesting that the in vivo phosphorylation pattern of FSV-R(950) PISO^^'1^ is similar to other FSV P 1 3 0 g a g - f p s proteins which have mutations at residue 1073. Since tyrosine-1073 is normally the major site of tyrosine phosphorylation within P1306ag-fps both in vitro (autophosphorylation) and in vivo, the lack of phosphorylation at tyrosine-1073 within FSV-R(950) P 1 3 0 g a g - f p s is consistent with i ts inability to undergo autophos-phorylation or to phosphorylate the exogenous substrate enolase in vitro. P 1 3 0 g a g - f p s isolated from FSV transformed cells is phos-phorylated not only at tyrosine, but also at serine and threonine res i -dues. This suggests that other cellular kinases are also involved in cellular transformation by FSV. Either the cyclic-nucleotide-dependent protein kinases or protein kinase C may be responsible for the phosphorylation of the serine and threonine residues. Although i t had been assumed that phosphotyrosine was the product of the tyrosine kinase activity intrinsic to the FSV transforming protein, phosphoamino - 195 -acid analysis and tryptic phosphopeptide mapping of FSV-R(950) P13Qgag-fps indicated that this mutant protein was phosphorylated at tyrosine residues in intact cel ls. The phosphorylation of tyrosine residues within FSV-R(950) P 1 3 0 g a g _ f p s , which is devoid of tyro-sine protein kinase activity, suggests that this protein may be phos-phorylated by a cellular protein kinase specific for tyrosine residues. On the other hand, the possibility exists that the tyrosine-phosphorylation detected in the FSV-R(950) P^Cp^'^ may be the product of a unique type of autophosphorylation. - 196 -CHAPTER 7 7.0 SUMMARY The transforming protein of FSV has an intrinsic tyrosine specific protein kinase activity and is i tself phosphorylated at multi-ple tyrosine and serine residues. Thus, phosphorylation of the FSV transforming protein is complex and may well affect i ts activity and function. Since the kinase and transforming activities of FSV P 1 4 0 g a g _ f p s are related, I have investigated the phosphorylation of the FSV transforming protein in detail. The major fps-specific phosphorylation sites of FSV P 1 4 0 g a g _ f p s , which include two phosphotyrosine residues and a phosphoserine residue, have been localized to the C-terminal region of the protein, which contains the kinase domain. Comparative tryptic phosphopeptide analysis has indicated that the phosphotyrosine residues are contained within a region that is highly conserved between the tranforming proteins of different FSV variants. This suggests that these phosphorylation sites are important for the activity of the protein. The relationship between the phosphorylation of the FSV transforming protein and i t s enzymatic and biological activities has been investigated. The^  strategy involved mutations at the major tyro-sine phosphorylation site within FSV PISO 5 3 - 6 "^ 8 , tyrosine-1073. Oligonucleotide-directed mutagenesis was used to change the codon for - 197 -tyrosine-1073 to a codon for phenylalanine, serine, threonine or glycine. These amino acid substitutions at position 1073 within P 1 3 0 g a g - f p s allowed evaluation of the effect of phosphorylation on the protein's activity and function. In addition the specificity of phosphorylation was determined by examining the serine and threonine replacements at residue 1073. A l l of the FSV-tyrosine-1073 mutant genomes transformed rat-2 cells and induced cells of nearly identical morphological phenotype; however, only after a long latent period. In addition these different mutant transformed cells expressed p l 3 0 gag- fps proteins with reduced kinase activities. The similar effects of these mutations was surprising considering that the R-groups of the substituting amino acids are quite different. The results obtained with the different mutations imply that the important characteristic of tyrosine-1073 is i t s abil ity to become phosphorylated. Therefore, given the caveat that mutations at residue 1073 do not have a global effect on protein con-formation, I have interpreted the reduced kinase activity and trans-forming potential exhibited by the mutant proteins to be a direct consequence of a lack of phosphorylation at residue 1073. The fact that neither serine nor threonine were phosphory-lated when placed at a tyrosine kinase recognition site demonstrates the specificity of the p 1 3 0gag-fps protein kinase activity for - 198 -tyrosine residues. While i t seems probable that the amino acids surrounding tyrosine-1073 of wtFSV P 1 3 0 g a g ~ f p s are important in targeting the kinase to a particular residue, i t is apparent that there i s a strict requirement for tyrosine at the site of phosphorylation. Amino acid substitutions at tyrosine-1073 of P l 3 0 g a g _ f P s did not completely abolish i ts activity. This suggests that phosphory-lation of tyrosine-1073 may not have a crucial function in catalysis, but may play a regulatory role. However, the results do not exclude the possibility that some activity of FSV P 1 3 0 g a g - f P s other than tyrosine phosphorylation is involved in i ts transforming abi l i ty . Although, these data strongly suggest that tyrosine phosphorylation of PISO^^-^P8 can modulate i t s activity and strengthen the case for the involvement of tyrosine phosphorylation in transformation by FSV. Mutations within the putative ATP-binding site of P 1 3 0 g a g ~ f p s at lysine-950 destroy both i ts kinase and transforming activit ies. These results support the idea that the tyrosine kinase activity intrinsic to p i 3 0 g a g - f P s i s essential for i t s tranforming function. A conservative amino acid change to arginine at residue 950 allowed for the stable expression of the mutant protein. Tryptic phos-phopeptide mapping and phosphoamino acid analysis of the mutant protein indicated that i t was not' phosphorylated at tyrosine-1073, but was phosphorylated at a second tyrosine site previously identified in wt P130 g a g ~ f P s as a site exclusively phosphorlyated in vivo. The - 199 -phosphorylation of tyrosine residues within a mutant protein devoid of intrinsic tyrosine protein kinase activity suggested that FSV p l 3 0 gag- fps may be a target for phosphorylation by a cellular protein kinase specific for tyrosine residues. The activity and function of the FSV transforming protein may be subject to modulation by a number of cellular kinases, each with different amino acid substrate specificities. The possible participation of a number of cellular kinases during FSV transformation is consistent with many investigations to date which reveal that cellular transformation by FSV occurs by a com-plex and as yet undefined mechanism. 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