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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 t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of The U n i v e r s i t y of B r i t i s h Col 1956 Main Mall Vancouver, Canada V6T 1Y3 D  a  t  e  ^)lJLfoJL.  U  s  l<jf&  ABSTRACT  The phosphorylation of the Fujinami sarcoma virus transforming protein (FSV P 1 4 0  g a g - f p s  its  protein  tyrosine  function.  specific The sites  of  )  is complex, reversible and affects kinase activity  phosphorylation within  and transforming FSV  pi40 S~ P ga  f  s  have been localized to various regions of the protein using partial proteolysis. The two major phosphotyrosine residues and a major phosphoserine 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 P130  g a g - f p s  phosphorylation.  Tyrosine-1073  was  mutated  to  of 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 strict tyrosine.  specificity  for  A l l of the FSV tyrosine-1073 mutants had depressed enzymatic (ii)  and oncogenic capacities. These data indicate that tyrosine phosphorylation stimulates the biochemical and biological activities of FSV PISO^ ^ ^^ 8  -  and suggest  8  that  tyrosine  phosphorylation modulates  protein function. Mutations PISO^ ^ ^ 8  -  within  the  putative  ATP-binding  site  of  at lysine-950 destroy both i t s kinase and transforming  8  activities, supporting the idea that the tyrosine kinase activity intrinsic  to  function.  P130^ ^~^P a  s  is  essential  for  its  transforming  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 by cellular 1  tyrosine specific protein kinases.  (iii)  TABLE OF CONTENTS Page Abstract List of Tables List of Figures . . . . . List of Abbreviations Acknowledgments Dedication  . . . . . . .  ii ix x xiii xvi xvii  CHAPTER 1 1.0  Introduction  1  1.1 1.2 1.3 1.4  1 1 2 3 3 4 5 6 7  1.5 1.6  1.7 1.8  1.9 1.10  Classification of Retroviruses Structure of Retroviruses Replication of Retroviruses Oncogenic Retroviruses 1.4.1 Onc+ Oncogenic Retroviruses 1.4.1.1 Transduction of Cellular Oncogenes . . 1.4.2 One- Oncogenic Retroviruses One and Proto-onc genes Retroviral Oncogene Products 1.6.1 Tyrosine Kinase-Negative Class of Oncogene Proteins 1.6.2 Tyrosine Protein Kinase-Positive and KinaseRelated Class of Oncogene Proteins Cellular Tyrosine Protein Kinases Cellular Substrates of Tyrosine Kinases 1.8.1 Indirect Cellular Substrates of Tyrosine Kinases Characteristics of Tyrosine Protein Kinases Phosphorylation of Tyrosine Kinases - A Possible Role in Regulating Activity 1.10.1 Tyrosine Phosphorylation and the Regulation of Kinase Activity 1.10.2 Serine and Threonine Phosphorylation and the Regulation of Kinase Activity (iv)  8 10 16 18 25 26 29 29 31  Page 1.11 Fujinami Avian Sarcoma Virus . . . . 1.11.1 The FSV Genome 1.11.2 Variant Strains of FSV 1.11.3 The Relationship of FSV to Other Oncogenic Viruses 1.11.4 The FSV Encoded Transforming Protein . . . . 1.11.5 Cellular Location of the FSV Transforming Protein 1.11.6 The Normal Cellular fps Homologue 1.11.7 Structure of the FSV Gene Product 1.12 Purpose and Experimental Approach  33 34 35 35 37 39 40 41 44  CHAPTER 2 2.0 Materials and Methods 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12  47  Cells and Viruses Radiolabelling of Cells Immunoprecipitation Immune Complex Kinase Reaction SDS-Polyacrylamide Gel Electrophoresis Partial Proteolytic Cleavage with pl5 and V8 Protease Analysis of Tryptic Peptides Analysis of Phosphoamino Acids Transfection of DNA into Rat-2 Cells Oligonucleotide-directed Mutagenesis Synthesis of p-Fluorosulfonylbenzoyl-5 -Adenosine . . Reaction of FSBA with P140g fi~ P 1  a  (v)  f  s  47 48 49 49 50 52 53 56 58 60 62 62  Page CHAPTER 3 3.0 Mapping of Multiple Phosphorylation Sites Within the Structural and Catalytic Domains of the Fujinami Avian Sarcoma Virus Transforming Protein 3.1 Introduction 3.2 Results 3.2.1 Tryptic Phosphopeptides of Pl40gag-fps 3.2.2 Localization of Phosphorylation Sites on P140gag-fps 3.2.3 Phosphorylation of the Transforming Proteins of Different fps Viruses 3.3 Discussion  63 63 65 6 5  70 86 92  CHAPTER 4 4.0 Oligonucleotide-direct Mutagenesis of Fujinami Sarcoma Virus: Evidence the Tyrosine Phosphorylation of P130Sag-fps Modulates i t s 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 Ill 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 Shows a Strict Specificity for Tyrosine Residues g a g - f p s  133  5.1 Introduction 5.2 Results 5.2.1 Oligonucleotide-directed Mutagenesis of FSV. . . 5.2.2 Transforming Activity of Wild-type, Mutants and Their Revertant FSV DNAs 5.2.3 Expression, Structure and In Vitro Kinase Activities of wt and Mutant Proteins 5.2.4 Tyrosine Phosphorylation of wtFSV and Mutant FSV PlSOg^-fps from Transformed Cells 5.3 Discussion  133 135 135 140 143 150 155  CHAPTER 6 6.0 Site-directed Mutagenesis of Lysine-950 Within P 1 3 0 Eliminates Both Its Kinase Activity and Transforming Ability  g a g _ f p s  159  6.1 Introduction 6.2 Results 6.2.1 Inactivation of P140S S- P Kinase Activity by Treatment with FSBA 6.2.2 Site-directed Mutagenesis of Lysine-950 Within the Putative ATP-binding Site of PlSOg^-fP . . 6.2.3 Transfection of Rat-2 Cells with Wild-type, Mutant and Revertant FSV DNAs 6.2.4 Expression and Characterization of Mutant FSV-R(950) and FSV-G(950) P130e &- P Proteins. . a  f  s  8  a  (vii)  159 164  f  s  164 169 172 176  Page 6.2.5 Trans-phosphorylation of FSV-R(950) Pl30gag~fps by FSV P140S g~ P 6.3 Discussion a  f  s  184 190  CHAPTER 7 7.0 Summary  1 9 6  References  2  (viii)  °0  LIST OF TABLES Page 9  Table 1.1  Retroviral Oncogenes  Table 1.2  Avian Sarcoma Viruses, Their Cell-derived Sequence Inserts and Gene Products  36  Quantitation of the Kinase Activities of gag-fps proteins Encoded by Wild-type, Mutant and Revertant FSV DNAs  115  Table 4.1  P130  Tabel 4.2  Phosphoamino Acid Analysis of p gag-fps 130  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-AMPDependent Protein Kinase  13  Figure 3.1  Tryptic Phosphopeptide Analysis of FSV P i 4 0  67  Figure 3.2  Cleavage of FSV P i 4 0  Figure 3.3  Tryptic Peptide Analysis of pl5 Cleavage Fragments of FSV P140g S- P a  f  with pl5  g a g - f p s  g a g _ f p s  . . . .  73  75  s  Figure 3.4  Cleavage of FSV P140g €- P with V8 Protease. . .  Figure 3.5  Cleavage of [ S]methionine or P-labelled FSV PMO^g-fps with V8 Protease Tryptic Peptide Analysis of V8 Protease Cleavage Fragments of FSV P140S g- P Mapping V8 Protease Cleavage Fragments of FSV Pl gag-fps  85  Cleavage Sites for pl5 and V8 Protease on FSV P140 S- P  88  Tryptic Phosphopeptide Analysis of trFSV P140g ^- P , FSV P 1 3 0 and PRCII P105g S-  90  Figure 3.6  a  f  35  f  s  40  Figure 3.8  ga  Figure 3.9  f  a  s  f  78  32  a  Figure 3.7  s  s  80 83  g a g _ f p s  a  fps  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 Transfection with FSV DNAs  109  Analysis of Wild-type, Mutant and Revertant FSVTransformed Rat-2 Cells for FSV PlSOg^-fps Synthesis and Tyrosine-specific Protein Kinase Activity . . . . .  113  Figure 4.5  (x)  Page Figure 4.6  Tryptic Peptide Analysis of P 1 3 0 Encoded by Wild-type, Mutant and Revertant FSVs g a g - f p s  Figure 4.7  Phosphoamino Acid Analysis of P 1 3 0  Figure 4.8  Whole Cell Phosphoamino Acid Analysis  Figure 5.1  The Synthetic Oligonucleotides Used to Mutate the Codon for Tyrosine-1073 of FSV P 1 3 0 & . .  138  Partial DNA Sequencing of wtFSV and Mutant M13mpl0 FSV Inserts  139  gag  ~  118 123 127  ga  Figure 5.2  . . .  fps  fps  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 in Transformed Rat-2 Cells and Quantitation of Their Respective Tyrosine Protein Kinase Activities . .  Figure 5.5  g a g - f p s  145  Tryptic Phosphopeptide Analysis of wtFSV and Mutant FSV PlSOSag-^P Proteins 149 8  Figure 5.6  Tryptic Phosphopeptide Analysis of P I S O ^ ^ Encoded by Wild-type and Mutant FSVs -  8  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-Fluorosulfonylbenzoy1-5 -Adenosine (FSBA) and Adenosine 5'-triphosphate (ATP) 162 1  Figure 6.2 Figure 6.3  Inactivation of pl406 &- P Kinase Activity By Treatment with FSBA  166  The Synthetic Oligonucleotdies Used to Mutate the Codon for Lysine-950 of FSV P130£ g- P . .  171  Morphological Phenotypes of Rat-2 Cells Following Transfection with FSV DNAs  175  a  f  s  a  Figure 6.4  (xi)  f  s  Page Figure 6.5  Analysis of Wild-type and Mutant FSV Transfected Rat-2 Cells for FSV P130gag- P Synthesis and Tyrosine Protein Kinase Activity  178  Comparison of Tryptic Phosphopeptides From PlSOgaS-fps Encoded by Wild-type, Mutant FSV-S(1073) and Mutant FSV-R(950)  182  f  Figure 6.6  Figure 6.7  s  In Vitro Phosphorylation of FSV-R(950) P i 3 0 by FSV P l 4 0  g a g  ~  f p s  g a g - f p S  Figure 6.8  Tryptic Phosphopeptide Analysis of In Vitro Phosphorylated wtFSV P130£ g- and In Vitro Trans-phosphorylated FSV-R(950) PISOS^-^P . . . a  fps  8  (xii)  186 189  LIST OF ABBREVIATIONS  A ADP AEV AK AMP ATP APE ASV bp C cAPK CEFs cGPK CHO Ci cpm C-terminal dATP DMEM DMSO DNA EDTA EGF ESV F FAV FBS FSBA FSV G G x g A G°  adenine adenosine 5'-diphosphate Avian erythroblastosis virus adenylate kinase adenosine 5'-monophosphate adenosine 5'-triphosphate alanine-proline-glutamic acid Avian sarcoma virus base pair cytosine cyclic-AMP dependent protein kinase chicken embryo fibroblasts cyclic-GMP dependent protein kinase Chinese Hamster Ovary Curie counts per minute carboxy-terminal portion of a protein 2'-deoxyadenosine 5'-triphosphate Dulbecco's modified Eagle's medium dimethylsulfoxide deoxyribonucleic acid disodium ethylene diaminetetraacetic acid epidermal growth factor Esh sarcoma virus phenylalanine Fujinami associated virus fetal bovine serum p-fluorosulfonylbenzoyl-5'-adensoine Fujinami sarcoma virus guanine glycine times gravity free energy (xiii)  GA-FeSV Gly GMP gs~ HAT IGF1 K kb kbp kcal kd Km Lys M uCi ul mCi mM NAD NCP98 N-terminal PDGF PCS PMSF pTkl PRC II PRC IV R Rf RF RNA  Gardner-Arnstein feline sarcoma virus glycine guanosine 5'-monophosphate group specific antigen negative hypoxanthine-aminopterin-thymidine insulin-like growth factor-1 (somatomedin c) lysine kilobase kilobase pair kilocalories kilodaltons Michaelis-Menton constant lysine Molar micro-Curie microliter milli-Curie millimolar nicotinamide adenine dinucleotide normal avian cellular fps gene product amino-terminal portion of a protein platelet derived growth factor Phase Combining System scintillation cocktail phenylmethylsulfonylfluoride plasmid containing the Herpes Simplex-1 thymidine kinase gene Poultry Research Centre II sarcoma virus Poultry Research Centre IV sarcoma virus arginine relative mobility replicative form ribonucleic acid  (xiv)  RSV S SDS SDS-PAGE Ser ST-FeSV T T TBE TCA TEMED Thr TK TK~ TLC TNE TPA TPCK tr ts Tyr UR1 UR2 vol wt wt/vol X Y Y73  Rous sarcoma virus serine sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis serine Snyder-Theilen feline sarcoma virus threonine thymine Tris-Borate-EDTA buffer trichloroacetic acid N, N, N', N'-tetramethylethylenediamine threonine thymidine kinase thymidine kinase minus mutant thin layer cellulose Tris-NaCl-EDTA buffer phorbol tetradecanoate acetate L-(l-tosylamido-2-phenyl) ethyl chloromethyl ketone temperature resistant temperature sensitive tyrosine University of Rochester 1 sarcoma virus University of Rochester 2 sarcoma virus volume wild-type weight by volume an unspecified amino acid tyrosine Yamaguchi sarcoma virus  (XV)  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 c a l reading of this thesis. Financial support is gratefully National Cancer Institute of Canada.  (xvi)  acknowledged  from  the  I would like to  to dedicate  my parents,  whose constant me throughout  Doris love  this and  thesis David  and support  my student  years.  (xvii)  has  with  love  Weinmaster, sustained  - 1 -  CHAPTER 1 1 . 0 INTRODUCTION  1.1 Classification of Retroviruses  Retroviruses have been isolated from a wide variety of vertebrates, as well as invertebrates and have been grouped according to common morphological, biochemical and physical properties (for a complete review, see Teich, 1982).  The Retroviridae virus family includes  a l l viruses containing an RNA genome and an RNA-dependent DNA polymerase (Fenner, 1975).  The family i s 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 clinical 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 r a l 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 transcriptase, which has both RNA-dependent and DNA-dependent DNA polymerase activities.  Both of these activities are essential to their  mode of viral replication which involves double-stranded DNA intermediates.  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  Swanstrom, 1982).  In the integrated state they can be transcribed like  other cellular genes.  (proviral forms)  (Varmus and  Thus, a unique feature of retroviruses i s 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 ability to transform cells.  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. Acquisition of viral transforming genes usually involves the loss of viral genes required for replication.  As a result, most of the viruses in  this class are replication defective and can only be grown in the presence of a helper virus; however, Rous sarcoma virus (RSV) i s 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 i s 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. homologous cellular genes, known as c-oncogenes (c-onc's)  The  (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 a l . , 1983; Varmus, 1984).  It  i s 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  Varmus, 1984).  a  retroviral  vector  (Bishop,  1983;  Duesberg,  1983;  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 i n v i r a l  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 i n tissue culture (Teich, et a l . ,  1982).  Evidence suggests that  tumors which arise  following infection with v-onc~ retroviruses contain mutant  cellular  oncogenes that have been activated by proviral  (Varmus,  1982b).  insertions  Integration of a provirus into the host genome i s potentially  mutagenic i f integration disrupts a v i t a l region of the host genome (Bishop, 1983).  Indeed, i t i s generally thought that such insertion  mutations are primary events i n tumorgenesis and their effect i s to stimulate expression of a cellular gene (c-onc) through the strong v i r a l promoter or enhancer element present within the proviral terminal repeat  (LTR) (Hayward,  et a l . ,  1981; Neel,  long  et a l . , 1981;  Payne, et a l . , 1981; Payne, et a l . , 1982; Fung, et a l . , 1983; Cuypers, et a l . , 1984; Nusse, et a l . , 1984).  1.5 One and Proto-onc genes  The retroviral oncogenes are transduced, multiply mutated, and highly 1984).  tumorigenic  forms of cellular  proto-oncogenes  (Varmus,  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 a l . , 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 demonstrate 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 r a l 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 counterparts.  1.6 Retroviral Oncogene Products  Extensive genetic evidence indicates that the protein product of a single RNA tumor virus gene i s generally responsible for the malignant transformation of Duesberg, 1983; Varmus, 1984).  virus  infected  cells  (Bishop, 1983;  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 c e 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; Alitalo, 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 Gene Product v-src p60  Viral Origin  src  Rous sarcoma virus  yes  Y73 avian sarcoma virus  fgr  Gardner-Rasheed P70 feline sarcoma virus gag-fps Fujinami sarcoma virus  fps  Eag  Cellular Horaologue p60  C_SrC  yeS  P90 "  Tyrosine protein kinase Tyrosine protein kinase  ga8_fgr  pl40  Activity  Tyrosine protein kinase C  fpS  p98 -  Tyrosine protein kinase  Abelson murine P120 leukemia virus p^gag-ros UR2 avian sarcoma virus  pi,50c-abl Tyrosine protein kinase  fes  Snyder-Theilen P85 feline sarcoma virus  p92  erb-B  erb-B Avian erythroblastosis virus gp65  fms  McDonough feline sarcoma virus  mil  MH2 avian virus  raf  3611 murine sarcoma virus  Potential serine/ threonine protein kinase  mos  mos Moloney murine sarcoma virus p37  Potential protein kinase  sis  sis Simian sarcoma p28 virus  Ha-ras  Harvey marine sarcoma virus  Kl-ras  .v-K-ras Kirsten marine P„ 21 sarcoma virus  abl ros  Wrabl  foe  FBJ murine osteosarcoma virus  myc  Avian myelc— cytomatosis virus K29 Avian myeloblastosis virus  myb  gag_feS  Tyrosine protein kinase Tyrosine protein kinase  Truncated Potential tyroEGF recep- sine protein tor kinase Potential tyrosine protein kinase  gag^nil  Potential serine/ threonine protein kinase  pl00  PDGF B-chain  „1 v-H-ras p2  fOS  p55  gag-myc  pll0  0Vb  p48  PDGF agonist GTP binding  ^jC-K-ras  GTP binding Possible DNA binding protein Binds DNA Possible DNA binding protein  The table l i s t s 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 homology corresponds to the kinase domain.  strongest  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  p60  src  that carries the kinase activity (Levinson, et a l . , 1981; Brugge and Darrow,  1984).  Using the amino acid sequence of p 6 0  src  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 i s thought to specify a functional domain that i s essential for the tyrosine protein kinase and  cellular  transforming  Parsons, 1984).  activities  of  p60  src  (Byrant  and  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 r s 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 . src  of p 6 0  src  The analysis revealed that residues 259 to 485  have 22% sequence identity with residues 38-258 of the  catalytic subunit of cAPK. maximal  amino acid  Alignment of the two sequences to obtain  identity  aligns  lysine-71 in the catalytic subunit.  lysine-295  in  p60  src  with  Both of these conserved lysine  -  Figure 1 . 1  12  -  Amino acid sequences from within the protein-kinase domain of 1 2 oncogene products and the cyclic-AMPdependent protein kinase (cAPK) are arranged to show their similarity.  A stretch in the middle of each  sequence has been omitted; omitted i s indicated.  the number of  subunits  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, Tyrosine. text. text.  Threonine;  V,  Valine;  W, Tryptophan;  Y,  The sequences blocked are discussed in the  References for the sequence data are cited in the  .VAI  K  TLKP  — 83  VHRDLRAANILVGENL  .. VCKVADFGLARLIEDNE  Y  TARQGAKF..PIKWT  APE  A  VA[  K  TLKL  — 83  IHRDLRAANILVGDNl  ...VCKIADFGLARLIEDNE  Y TARQGAKF..PIAWT  APE  A  K  TLKP  — 83  IHROLRAANILVGERL  ,, VCK[ADFGLARLIEDNE  Y NPRQGAKF..PIKWT  APE  A  VAV  K  TLKE  — 84  IHRDLAARNCLVGENH  Y  APE  S  ,VAV  K  SCRE  — 85  IHRDLAARNCLVTEKN  ...TLKISOFGMSRQEEOGV  Y ASTGGMKQI.PVKWT  APE A  EVFSGRLRADNTL  K  SCRE  — 85  IHRDLAARNCLVTEKN  . . . VLKISDFGMSREEADGV Y AASGGLRLV.PVKWT  APE A  GSGAFG  EVYEGTALDILADGSGESRVAV  K  TLKR  — 91  IHROLAARNCLVSEKQYGSCSRVVKIGDFGLARDIYKND  Y  YRKRGEGLL.PVRWM  APE  S  VI  GSGAFG  TI YKGLWIPEGEK. . V T I P V A I  K  ELRE  — 84  VHRDLAARNVLVKTPQ  K  EYHAEGGKV.PIKWM  ALE  S  fms  TI  GTGAFG  KVVEATAFGLGKED . A V L K V A V  K  MLKS  - 155 IliRDVAARNVLLTSGR  mi 1  RI  GSGSFG  TVYKGKWHGD  ,VAV  K  ILKV  — 85  IHRDMKSNNIFLHEGL ,  E  raf  RI  GSGSFG  TVYKGKWHGD  VAV  K  ILKV  — 85  IHRDMKSNNIFLHEGL, .  G SQQVEQPTG.SVLWM  mos  RL  GSGGFG  SVYKATYHG  K  QVNK - 100  CAPK  TL  GTGSFG  RVMLVKHMETGNH  K  ILDK — 86  src  KL  GQGCFG  EVWMGTW'JDTTR  yes  KL  GQGCFG  EVWMGTWNGTTK  fgr  RL  GTGCFG  DVWLGMWNGSTK  abl  Kl  GGGQYG  EVYEGVWKKYSLT  .,,  fps  RI  GRGNFG  EVFSGRLRADNTP  ..,  fes  01  GRGNFG  ros  LL  erb-B  ,.  ..,  YAM  ,  LVKVAOFGLSRLMTGDT  ,  IYRDLKPENLLIDQQG, .  VAKIGDFGLARDIMNDS  TAHAGAKF..PIKWT  N YIVKGNARL.PVKWH SQQVEQPTG.SILWM  APE s APE  V  APE V  G RQASPPHIGGTYTHQ APE  I  W T.LCGT  I  PE.YL  APE  1 CO  1  - 14 -  residues have been shewn to react specifically with the ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine Kamps, et a l . , 1984).  (FSBA)  (Zoller,  et a l . , 1981  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  src  tyrosine kinase probably orient  residue in a similar conformation.  the homologous lysine  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 functional 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 varying degrees of homology to p 6 0  src  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 i s 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, 1983).  et a l . ,  It i s interesting that site-specific mutagenesis of lysine-121  in mos, which i s homologous to lysine-295 in p 6 0 , destroys the src  transforming activity of mos (M. Hannick and D.J. Donoghue, personal communication). The meaning of this kinship i s 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 r s t tyrosine kinase identified in normal  cells was the cellular homologue of p 6 0  src  (Collett,  Oppermann, et a l . , 1979; Col lett, et a l . , 1979b).  et a l . , 1978;  Since then a number  of normal cellular homologues of the oncogenic tyrosine kinases have been identified.  Their differential expression in various cell 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 c e 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 regulation of normal cellular growth i s 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 tyrosine specific protein kinase activity.  The binding of the respective  - 17 growth factors to their receptors results not only in the phosphorylation 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 stimulated 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 tyrosine, 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 c e l 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 accompanies 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 phosphotyrosine levels (Cooper and Hunter, 1981a), which indicates that the phosphorylation of cellular target proteins may be involved in the mechanism of transformation. regulatory mechanisms.  Inappropriate phosphorylation of key  proteins could disrupt  normal cellular growth  control  The detection of tyrosine kinase activity associated with  certain growth factor receptors and the enhancement of cellular phosphotyrosine 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 c e l l growth.  It  may be that some oncogenic proteins have the same activity and overlapping protein specificity as some growth factor receptors, no longer subject to the same regulation.  but are  - 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 i s 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) i s apparently a truncated  version of the EGF receptor (Downward, et a l . , 1984a; Ullrich, 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 substrate proteins for tyrosine kinases i s obviously important, since they may be involved in the control of cellular growth in both normal and malignant c e l l s .  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 surprising considering the proposed common ancestry of the src family catal y t i c 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 i s a multifaceted process which alters the cells in a number of ways, including loss of contact inhibition, abnormal glucose metabolism and alterations in c e l 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 c e l 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 c e l l  shape.  Since p 6 0  src  is  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  p60  src  may  interfere  with  its  function and result in disorganization of the actin-containing microfilaments leading to an alteration in c e l 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 c e 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 cell is phosphorylated (Sefton, et a l . , 1981).  In addition, quantitative changes in both fibronectin  and vinculin may contribute to the changes in c e l l shape (Olden and Yamada, 1977; Iwashita, et a l . , 1983).  - 21 -  A well known characteristic of tumor cells i s 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, like 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 cells.  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 phosphorylated at tyrosine  (Cooper, et a l . , 1983b; Cooper,  et a l , 1984a)  Perhaps the enhanced rate of glycolysis seen with transformed cells i s 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 c e l 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 r e s 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) i s the most extensively characterized tyrosine kinase substrate,  being the f i r s t  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 i s 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). gpggerb-B with p 6 0  The f r o m  src  36K  ^  phosphoprotein  co-immunoprecipitates  transformed cells (Gilmore,  with  et a l . , 1985) and  from RSV transformed cells (Dehazya and Martin, 1985),  suggesting that these complexes may represent stable enzyme-substrate associations. However, p36 i s 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 protein kinases (Cooper and Hunter, 1983b). The phosphorylation of p36 i s 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 phosphorylation (Nakamura and Weber, 1982; Cooper, et a l . , 1983b).  Both the  phosphorylated and unphosphorylated forms of p36 are found predominately in the plasma membrane, where i t may perform a structural function (Cooper and Hunter, 1982; Courtneidge, et a l . , 1983; Greenberg and Eldeman, 1983; Radke, et a l . , 1983).  Even though p36 i s abundant in  fibroblasts, i t i s not found in a l l cells (Cooper and Hunter, 1983b).  - 23 -  A 42,000 dalton phosphoprotein i s detected in chick cells, 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 c e l l division.  Unlike that found in RSV transformed chick cells,  treatment of chick cells with mitogenic agents results in very few phosphorylated proteins, although p42 i s 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  its  identity and function remain an enigma. Along with the 36K and 42K proteins, an 81,000 dalton protein (p81) i s also phosphorylated at tyrosine following treatment of A431 cells with EGF (Hunter and Cooper, 1981c).  The same protein i s 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 cells.  The physiological significance of this EGF-induced phosphoryla-  tion i s questionable, since the EGF dose required to obtain phosphorylation of pSl leads to an inhibition of the growth of A431 cells (Gill 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 cells, p60  farc  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, pl05  et a l . ,  gag-fps  et a l . ,  1981).  (Adkins,  The  et a l . ,  1982) and P140 g~ P ga  f  s  oncogenic 1982),  (Pawson,  tyrosine  (Lipsich,  m)^^yes  et a l . ,  kinases  1981) are  also  found in a complex with p50 and p89. It i s thought that the complex i s involved in shuttling p 6 0  src  from i t s site of synthesis on soluble  polysomes to the plasma membrane.  p50 i s phosphorylated at a single  tyrosine residue (Hunter and Sefton, 1980; Oppermann, et a l . , 1981) in transformed cells, but not in normal cells (Brugge and Darrow, 1982; Gilmore, et a l . , 1982). Whether the tyrosine phosphorylation of p50 i s fortuitous or functional, i t seems likley that p50 i s a substrate for the tyrosine protein kinase with which i t i s associated. Despite the identification of several cellular proteins containing high levels of phosphotyrosine in virally transformed cells and cells treated with growth factors, there i s no direct proof that the phosphorylation of these proteins i s necessary or sufficient for transformation 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 i s crucial for transformation. 1.8.1  Indirect Cellular Substrates of Tyrosine Kinases  Although much attention has been focused on tyrosine phosphorylation, analysis of specific proteins, such as the ribosomal protein S6, reveals that protein phosphorylation at serine residues i s 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 (NilsenHamilton,  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 i s 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 c r i t i c a l for transformation as protein phosphorylation.  The possi-  b i l i t y remains that the src family of transforming proteins may a c 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 i s a recently recognized protein modification f i r s t identified in a number of viral transforming proteins (Eckhart,  et a l . , 1979; Collett,  Sefton, 1980; and Witte, et al •, 1980).  et a l . , 1979a; Hunter and Although i t has been assumed  that the phosphotransferase activity i s 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,  etal.,  1979) has since been shown to be the property  of  p60  c 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 Nomenclature 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:  protein O-phosphotyrosine).  ATP + protein tyrosine = ADP +  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 . phosphate donor,  Although ATP i s the preferred  dATP and GTP can be substituted in some cases  (Richert, et a l . , 1982).  Interestingly, the enzymatic properties of  the UR2 transforming protein,  -1  08  pesgag " ,  are  distinctive  from  those of the other avian sarcoma virus protein kinases in cation preference, 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 i s 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 i s also important for the recognition of phosphorylation sites by these enzymes. The protein bound tyrosine phosphate i s a high energy linkage.  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 i s important since hydrolysis or formation of such a high energy tyrosine phosphate bond in proteins could conceivably bring about a conformational 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 i s a reversible event (Fukami and Lipmann, 1983).  The dephosphorylation of  phosphotyrosine residues i s 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 . (Collett, s r c  and Gordon, 1984).  et a l . , 1984; Brown  This enhanced phosphorylation was due to an  increase in tyrosine rather than serine phosphorylation of p 6 0 , src  and could be correlated with a significant increase in i t s tyrosine kinase activity.  The  increase  in  tyrosine  phosphorylation  of  - 30 -  rj60 ^ br  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 1983).  of  ATP-Mg *  (Purchio,  2  et a l . ,  1983; Collett,  et a l . ,  Only when lysis was conducted at high ATP concentrations were  the new sites phosphorylated.  Perhaps this phosphorylation when s u f f i -  ciently extensive exerts an allosteric effect on p 6 0  src  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  src  that are only  detected under conditions which inhibit phosphotyrosine specific phosphatases.  Alternatively, vanadate may directly or indirectly activate  other kinases which results in the phosphorylation of these recently identified phosphotyrosine sites in p 6 0 . src  The vanadate experiments appear to suggest that the level of phosphorylation of tyrosine protein kinases and their cellular substrates w i l l depend upon the phosphotyrosine specific phosphatases in the c e l l .  Since reversible phosphorylation i s thought to be a major  mechanism for regulating protein function, i t seems reasonable to conclude 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  it  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 tyrosine 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 cells, 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  src  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  src  is  unaltered in both i t s biochemical and biological activities (Cross and Hanafusa, 1983), suggesting that this site of phosphorylation i s not essential for the activity of the protein. cAPK increases the ability  of p 6 0  src  However, phosphorylation by to phosphorylate casein at  tyrosine residues (Sefton and Hunter, 1984).  Treatment of RSV- trans-  formed Chinese hamster ovary cells  with cholera toxin  8-BromocAMP  also  stimulates  the  (CHO)  phosphorylation. of  p60  src  or at  serine residues, concomitant with an apparent increase in kinase a c t i vity (Roth, et a l . , 1983).  Interestingly,  p60  src  expressed and  synthesized in E. coli i s not phosphorylated, yet i t possesses about 10% of  the kinase activity  assayed for  cells (Gilmer and Erikson, 1981).  p60  src  from eukaryotic  These data suggest that phosphoryla-  tion i s 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 t s receptor (Magun, et a l . , 1980). serine/threonine  These treatments activate the cAMP dependent 2+ 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 phosphorylation on serine and/or threonine residues.  1.11 Fujinami Avian Sarcoma Virus  Fujinami sarcoma virus (FSV) i s an acutely oncogenic retrovirus, which was isolated from a naturally occurring chicken fibrosarcoma 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 ability of FSV i s 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 nontransforming 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 is:  5' -Upj-leader-gag-f ps-C-Ug-R-3 . 1  The 5» 1.0 kb of the FSV  genome contains a 21 base pair (bp) sequence that i s repeated at both termini of the viral RNA (R), an 80 bp unique sequence (U ) and the 5  leader sequence together with part of the FAV gag gene, which encodes the precursor virion core protein  The internal 3.0 kb i s  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  (Pi40  gag-f  P ) s  or  (P130 ~ P ), depending upon the strain used. gag  f  s  130,000  daltons  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, the transforming protein of FSV p gag-fps throughout this thesis.  as  either  I w i l l refer to pi40  g a g _ f  P  s  or  130  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 cell 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  Rous sarcoma virus (RSV)  Class  Cell-derived Sequences  TransformationSpecific Protein  I  src  p60  II  fps  Pl^gag-fps  src  Avian sarcoma virus B77 Fujinami sarcoma virus (FSV) Avian sarcoma virus PRCII  piOs^g-fps  Avian sarcoma virus PRCIV  pi70g g- PS  Avian sarcoma virus UR1  Pl gagrfps  Avian sarcoma virus 16L  P142g g- P  Avian sarcoma virus Y73  50  a  f  III  yes  P9 g g yes pse^g-yes  IV  ros  P6 gag-yes  Esh sarcoma virus (ESV) Avian sarcoma virus UR2  f  a  0  8  a  -  s  - 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 i s related  structurally and functionally to at least six groups of acutely oncogenic 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 equivalent or related sequences from genomes of two distantly related species. 1.11.4 The FSV Encoded Transforming Protein  The 4.5 kb FSV RNA i s 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). is  pi4o£ g" P a  f  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 transforming 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 P140  gag  ~  fps  is  and biochemical data have  the transforming protein of  FSV  suggested (Pawson,  that  et a l . ,  1980; Pawson, et a l . , 1981; Lee, et a l . , 1981; Hanafusa, et a l . , 1981; Lee, et a l . , 1982).  pi40  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 vitro kinase reaction P 1 4 0  g a g - f p s  In an  can phosphorylate both i t s e l f  and exogenous substrates specifically at tyrosine residues.  Cells  transformed by FSV have enhanced phosphotyrosine levels compared with nontransformed cells,  suggesting that P l 4 0  g a g - f p s  functions  tyrosine kinase in vivo.  Studies with temperature sensitive  mutants of  that  FSV indicate  phosphorylation  of  Pl40  as a  g a g _ f p s  (ts) is  necessary for initiation and maintenance of the transformed state of the c e l 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 c e l l phosphotyrosine levels, as well as phosphorylation of the transforming protein and i t s 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 phosphorylation of key cellular proteins regulating cell morphology, metabolism and growth could bring about the myriad of changes associated with transformation by FSV. location  of  One would anticipate that the intracellular  pi-iO - "* ^ 23 6  1  8  would  influence  the  structures,  sub-  strates, and regulators with which i t must interact to induce the process  of  P140 ~-*gag  ps  cell  transformation.  A  substantial  fraction  of  i s associated with the plasma membrane or cytoskeletal  structures in FSV-infected cells; however, this association i s salt sensitive P140  gag  ~  (Feldman, fps  et a l . ,  1983; Moss,  encoded by a ts  mutant of  et al•, FSV  1984).  The  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 c r i t i c a l to i t s transforming capacity.  The identity and intracellular locations of the physiologically  significant  targets  of p i 4 0  gag  ~^  ps  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 ~-^P , gag  a  S  98,000 dalton protein (NCP98), was identified in uninfected chicken tissue (Mathey-Prevot, et a l . , 1982). region  of  pi40  gag  ~P f  s  and has  NCP98 i s homologous to the fps  an associated  tyrosine  specific  kinase activity. Curiously, NCP98 isolated from cells does not appear to  be phosphorylated  at  tyrosine  residues,  unlike  pi40  g a g _ f  P . s  NCP98 i s 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  its  homologous transforming pi40g S~ P a  f  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 transforming  protein  of  the  recombinant  virus  F36  (P92 P ) f  s  lacks  gag  sequences (Foster and Hanafusa, 1983), and is also recovered in the  - 41 particulate  fraction  like  P140  g a g - f p s  (Young and Martin,  1984).  The results suggest that an alteration in the fps sequences is responsible for the different cellular localization between the FSV transforming protein and i t s normal cellular homologue, and that association with membrane or  cytoskeletal structures  is  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 i s encoded by sequences derived from FAV; (2) the C-terminal region (residues 888-1182), which i s homologous to the C-terminal domain of p 6 0 ; src  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 i s not entirely understood.  The gag protein does not appear to  influence intracellular locations of the gag-one fusion protein, since p60 , src  which contains no gag protein sequences, and  -  pgo^g ^  which does, are found at very similar intracellular locations. addition,  p55 , fos  0  pnogag-W ,  p  48 y m  b  and  6 8  In c  p66^  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, viral 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, tumorinducing ability 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 transforming proteins suggests that the association i s 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 i s 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 ability to induce B-cell lymphomas in mice but the virus can s t i l l transform fibroblasts in cell 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 t s interaction with specific cellular substrates. The C-terminal region of  P130  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  phosphotransferase activity.  possesses  the  catalytic  site  for  Several lines of evidence suggest that  the structural and functional properties of the catalytic domain may be shared among different  tyrosine  proteolytic  fragment  of  (Weinmaster,  et a l . , 1983),  kinases.  pi4oS 6 P a  -f  s  First,  possess  a  kinase  as does the corresponding  pgQsrc (Levinson, et a l . , 1981; Brugge and Darrow, EGF receptor (Basu, et a l . , 1984).  C-terminal activity region  of  1984) and the  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), eliminates  both  the  kinase  and  transforming  activities ' of  p60 , src  confirming the importance of the tyrosine kinase domain in transformation (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. transforming  The phosphorylation of tyrosine residues within the FSV protein  probably  results  from  autophosphorylation  occurring either in trans (Mathey-Prevot, et a l . , 1984), cis, or both.  - 45 -  However, the identity of the kinases involved in the serine and threonine phosphorylations i s unknown.  Several lines of evidence  suggest that various functional properties of tyrosine kinases may be regulated by phosphorylation  (section  regulation of the p i 4 0 ~ P  kinase activity i s an important step  gag  :f  s  1.10).  Understanding  the  in understanding the mechanism of transformation by FSV. In this study I have investigated the phosphorylation of FSV P140  gag  ~  fps  in detail to  establish the relationship between  vivo and in vitro phosphorylation  of  P140  multiple sites of phosphorylation on p i 4 0  g a g - f p s  g a g _ f p s  ,  .  in  To map the I have utilized  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 i s a major mechanism for the regulation of protein function (Krebs and Beavo, 1979). Therefore 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 pl30  gag-fps I  major  site  of  tyrosine  tyrosine-1073 (Chapter 4).  phosphorylaton  have mutated the  within  this  protein,  - 46 -  In order to understand more fully the biological role of -  PISO^B ^  and i t s kinase activity,  8  i t i s necessary to character-  ize certain biochemical properties of this protein.  One important  property of any protein kinase i s i t s substrate specificity.  There-  fore, I have investigated the specificity of the protein kinase i n t r i n 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. sic to PlSC "-*63,6  ps  If the kinase activity i n t r i n -  i s crucial for i t s transforming activity,  would predict that alterations in the P 1 3 0  gag  ~  fps  one  ATP-binding site  would eliminate not only i t s kinase activity, but also i t s transforming activity.  To test this hypothesis and to locate the ATP binding site  within the catalytic domain of pl30  gag-fps I  have mutated the  highly conserved lysine-950, which by homology with the src kinase i s 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 i d e n t i f i cation of certain structural and functional domains within the transforming 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. resistant  (tr)  derivative,  trFSV  (Lee,  et a l . ,  A temperature  1981) was  also  pseudotyped with FAV.  Both of these FSV strains encode a 140,000  dalton  g a g _ f p s  protein  (P140  ).  FSV  clone  12 isolated  from  a  different FSV stock by Hanafusa, et a l . , (1981) encodes a 130,000 dalton  protein  (P130  g a g - f p s  ).  PRCII  rescued  pheasant virus was obtained from G.S. Martin.  gs  with -  ring-neck  chicken embryo  fibroblasts (CEFs) were obtained from H & N Farms. Cells were infected with virus  (5 x 10  7  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 line, 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), penicillin 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; cals, Inc.)  ICN Pharmaceuti-  in 1.0 ml of phosphate-free DMEM containing 1-2% FBS.  After the labelling period the plate was transferred to ice, the radioactive medium removed, and the cells were washed two times with i c e cold phosphate-buffered saline (138 mM NaCl, 2.68 mM KC1, 1.5 mM KH2PO4,  8 mM Na HP0 [pH 7.2]) and then lysed in a total of 500 2  4  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. labelled with except  that  [ S]methionine  according  to  the cells were incubated with  a  Cells were  similar protocol,  [ 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). as described above.  The c e l l lysates were cleared and treated exactly  - 49 2.3  Immunoprecipitation Prespun cell 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 c e l 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 Immunoprecipitates  of  0,£,  P-labelled cells  (pH 8.0) - 0.1% NP40. 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 10 cells/ml from normal or transformed 5  cells were lysed in kinase lysis buffer (1.0% NP40 - 20 mM Tris-hydrochloride (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 MnCl - 20 mM 2  Tris-hydrochloride  (pH  7.5)  and  incubated  with  1-30  uCi  of  [^_ p]ATP (3,000 Ci/mmol; Amersham) in 35 ul of 10 mM MnCl 32  2  -  - 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 6.8),  10% [vol/vol]  glycerol,  .001% Bromophenol blue)  (pH  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). trations in the separating gel were as follows: chloride (pH 8.8) and 0.1% SDS.  The final concen0.375 M Tris-hydro-  The gels were polymerized chemically  by the addition of 0.1% by volume of N, N, N',N -tetamethylethylenedia1  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. P-labelled  proteins  were  either  After electrophoresis, gels of covered  with  Saran Wrap and  exposed to film (Kodak XAR-5) while wet at 4°C or fixed and stained by soaking overnight i n , 0.04% Coomassie Brilliant 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 distilled water.  The destained gels were dried onto 3MM  f i l t e r paper (Whatman) using a Hoefer slab gel drier. of  The sensitivity  P detection was increased by using an intensifying  screen  (DuPont; Lightning Plus) with XAR-5 film at -80°C (Laskey and Mills, 1977).  Gels of [ S]methionine-labelled proteins were impregnated 35  with En Hance (New England Nuclear Corp.) before drying and exposed 3  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-film 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 scintillation counting in the presence of PCS scintillant (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 electrophoresis.  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 r s 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. resumed at 3 watts.  Electrophoresis was then  Gels were fixed, stained, dried and treated as  described in section 2.5  2.7 Analysis of Tryptic Peptides  3 2  P  or  [ ^S]methionine 3  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 NH H00 , 0.1% 4  SDS).  3  The swollen pieces were crushed and homogenized with the flat  end of a disposable 5 ml syringe plunger with more buffer being added when necessary. was 5.0 ml.  Including washings, the final volume of buffer used  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 clinical 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 trichloroacetic 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 dryice/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 NH H00 4  3  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. OO  was detected by exposing the plates to XAR-5 film at -80°C with the aid of an intensifying screen (Laskey and Mills, 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  ^P-orthophosphate  u  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 (redistilled), at room temperature. The sample was vortexed (full 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 centrifugation 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. the plate was dried and then re-wetted with  After electrophoresis  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. qo 0/S  P-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 phosphoamino acids were lyophilized, separated by electrophoresis, identified 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 Sstl, 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 CaCl to an 2  equal volume of a solution containing 50 mM Hepes, 1.5 mM NaHP0 and 4  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 dexamethasone 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 experiment.  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 10 cells seeded in 4  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), isola-  tion of the 4.7 kbp FSV insert, cloning into the Sst 1 restriction site of M13mpl0 RF DNA, transformation of E. coli JM101 (D lacpro, SupE, thil,  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 oligonucleotide 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 AppliedBiosystems reagents.  Purification of  oligonucleotides  following  synthesis was carried out using a 20% polyacrylamide-7 M urea sequencing 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 oligonucleotide 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. c o l i DNA polymerase (large fragment) (BRL) and T4 DNA ligase (BRL) and was then used to transform competent E. coli 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 nitrocellulose (Schleicher and Schuell) and screened by hybridization with the mutagenic oligonucleotide which had been 5'-end-labelled with qo  ^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 t e r 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  gag  ~P f  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% glycerol.  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 final 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 Transforming 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 phosphorylated 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 c e 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 (P140 ~ ) gag  fps  or  a  130,000  dalton  protein  protein  (PISO ^"^ ), 89  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 al.,  1980; Lee, et a l . , 1980).  replication-competent virus  The nondefective gag gene of a  encodes a precursor  five virion core proteins (Vogt, et a l . , 1975). shown  that  P140 ~ P gag  f  possesses  s  an  (Pr76 ) gag  to the  It has previously been  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  immunoprecipitated with antiserum directed  s  against antigenic determinants in i t s gag or fps regions i s i t s e l f phosphorylated exclusively in vitro  with  Pawson,  et a l . ,  P140  gag  ~  fps  [ % - P]  ATP  32  1980;  isolated  at tyrosine and Mn  Pawson,  from  residues,  (Feldman,  2+  et a l . ,  transformed  after  1981). cells  et a l . , 1980; In  is  incubation  contrast,  phosphorylated  mainly at serine, tyrosine and possibly threonine residues (Pawson, et a l . , 1981).  However, the tryptic phosphopeptides of P 1 4 0  phosphorylated  in vitro  phosphorylated P 1 4 0  gag_f  P  are s  similar  to  those  from  g a g - f  P  s  in vivo-  (Pawson, et a l . , 1981).  The phosphorylation of the FSV transforming protein i s complex and may well affect i t s activity and function. activity  and transforming activity  of P 1 4 0  gag  ~  Since the kinase fps  are presumably  related, I have investigated the phosphorylation of FSV p i 4 0 in detail.  gag  ~P f  s  The aim of this study was to establish the relationship  between in vitro and in vivo phosphorylation of pi40 ~^P , gag  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~ P £  B  To identify the sites of phosphorylation in the FSV transforming protein, I trypsin  have analysed the phosphopeptides produced by  digestion of  Pl40  (ts) L5 strain of FSV.  g a g  ~  from the  f p s  temperature  sensitive  pi40 S~ P was isolated by immunoprecipiga  f  s  tation from FSV-transformed cells which was either metabolically 32 labelled with  P-orthophosphate or phosphorylated in vitro in the  immune complex with [ ^ - P ] 32  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  is  phosphorylated  exclusively  at  tyro-  sine residues in the in vitro immune complex kinase reaction (Feldman, et a l . , 1980; Pawson, et a l . , 1980). of  pi40  g a g  ~  f p s  phosphorylated  Tryptic phosphopeptide analysis  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 FSV  Pl40  P  g a g _ f  was  s  labelled  with  3 2  P  g a g  ~  f p s  .  in vivo op  by  incubation  isolated  by  of  transformed CEFs with  subsequent  Pj[  and  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  ~  was then digested  f p s  with trypsin and separated in two dimensions on thinlayer cellulose plates. origin.  An  indicates the sample  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: tsFSV p i 4 0 3 2  Pi  gag  ~P , f  phosphorylated  s  labelling);  B,  tsFSV  in vivo pi40  gag  A,  (18 hour  ~P f  phos-  s  qp  phorylated tsFSV  in vivo  pi40  gag  ~P f  (4  hour  Pj_  phosphorylated  s  labelling);  in vitro.  C,  Phos-  phoamino acid analysis of tryptic peptide from in vivolabelled  pi40  g a g  "P f  s  showed  that  spots  1,  3a  through 3c, 4, and 6 contain predominantly phosphotryosine, whereas spot 5 contains predominantly phosphoserine.  - 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 rela-  tively minor tryptic phosphopeptides 1 and 2 apparently represent d i s 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 t s intrinsic kinase activity with the residues actually phosphorylated  in  the  ^P-labelled  transformed  cell,  pi40  ts FSV L5-transformed  g a g - f p s  chicken  was  isolated  embryo  from  fibroblasts  (CEF) and subjected to tryptic phosphopeptide analysis (Figure 3.1A and B).  Secondary analysis of  pi40  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-phosphorylated P 1 4 0  g a g - f p s  .  A major new tryptic phosphopeptide (spot  4) which contains phosphotyrosine as i t s sole phosphoamino acid is present in the digest of in vivo-labelled P 1 4 0 of in vitro-phosphorylated p i 4 0  g a g - f p s  ments on tryptic digests of p i 4 0 S ~ ga  g a g - f p s  i s missing. fps  ,  and peptide 2 Mixing experi-  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 P140  gag-f  P  comigrate with  s  ted from  32  P-labelled,  those  found  in  FSV (FAV)-infected  FAV  Pr76  cells,  indicating  they represent normal sites of gag phosphorylation. P140gag-fps possesses a strongly  isola-  gag  labelled tryptic  that  In addition, phosphopeptide  containing phosphoserine (spot 5) which is not found in FAV P r 7 6  gag  and thus represents specific phosphorylation of a serine residue on P140  g a g  "  f p s  .  P140gag-fps i  These  results  indicate  that  ts FSV  L5  phosphorylated in transformed cells on three tyro-  s  sine residues (contained within tryptic peptides 1, 3a through 3c and 4), of which one (peptide 4) i s not phosphorylated in vitro, and on several serine residues of which at least one (peptide 5) i s specific to  P140  g a g - f p s  .  Two other  from in vivo-phosphorylated  FSV-specific  pi40 S~ P ga  f  s  tryptic  (peptides  phosphopeptides 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, i n d i cating that the majority of tryptic phosphopeptides have been separated.  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 g P a  -f  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 i s the sum of these spots), although i t i s difficult to know whether this reflects the actual steady-state levels in vivo. vitro-phosphorylated P 1 4 0  g a g  ~ , f p s  On in  spots 3a through 3c represent a  single major site of tyrosine phosphorylation, with tryptic phosphopeptides 1 and 2 comprising relatively minor phosphorylation sites. 3.2.2 Localization of Phosphorylation Sites on P l 4 0  g a g  "P f  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. FSV P l 4 0  g a g - f p s  By constructing such a proteolytic cleavage map of  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  gag  ~  fps  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). phosphorylation  sites  to  these  two  fragments,  To localize  pi40  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 immunoprecipitated protein.  Figure 3.2 shows that both the N-33K(pl5) and the  O120K(pl5)  fragments  pi gag-fps  a r e  40  of  in vivo-  labelled  with  and 3 2  in vitro-phosphorylated  P.  Phosphoamino  acid  analysis of the N-terminal 33K fragment of in vitro-phosphorylated P140 ing  g a g _ f p s  revealed only phosphotyrosine whereas the correspond-  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). phopeptide  analysis  P140  phosphorylated  g a g - f p s  of  the  N-terminal  in vivo  33K  yielded  the  Tryptic phos-  gag-fragment acidic  of  phospho-  tyrosine-containing tryptic peptide 1 in addition to phosphopeptides which comigrate with those of FAV P r 7 6 N-33K(pl5)  fragment  labelled only C-terminal P140  gag  ~  fps  at  of  tryptic  120K pl5 gives  (Figure 3.3A).  gag  in vitro-phosphorylated phosphopeptide  cleavage  fragment  Pl40  g a g  ~  The f p s  1 (Figure 3.3B). of  is The  in vivo-phosphorylated  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  gag  ~  fps  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 P r 7 6 . gag  However, the major sites of tyrosine  (peptides  - 72 -  Figure 3.2:  Cleavage Pl  of  40gag-fps  FSV  pi40  g a g  ~  labelled  rag  with  f p s  pl5.  in vivo  with  FSV 3 2  Pi  and isolated by immunoprecipitation with anti-pl9 serum or phosphorylated in an immune complex kinase reaction with [^- P]ATP. 32  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-polyacrylamide gel. phosphorylated uncleaved;  B,  Lanes: in vivo  A and B, tsFSV P l 4 0 (18  hour  cleaved; C and D,  labelling); tsFSV  Pl40  phosphorylated in vitro; C, uncleaved; D, cleaved.  g a g  ~  f p s  A, g a g - f p s  - 73 -  4i  PI  P140-H& m  D | — -«-120K  :  -*-33K  - 74 -  Figure 3.3:  Tryptic peptide analysis of pl5 cleavage fragments of FSV  pi40  P .  g a g _ f  s  3 2  P-  or  35  S-labelled  polypeptides were gel purified, digested with trypsin, and analysed by two-dimensional separation on thin-layer cellulose plates. follows:  Protein fragments analysed were as  A. N-33K(pl5) obtained by pl5 cleavage of  tsFSV P 1 4 0  g a g - f  P  phosphorylated  s  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  pi40  gag  ~P f  labelled  s  with  qc  [ S]methionine  in vivo;  OJ  pl5  cleavage  of  D,  tsFSV  C-120K(pl5) obtained by  pi40  gag  ~P f  s  phosphorylated  in vivo; E, O120K(pl5) obtained by pl5 cleavage of tsFSV  P140  gag  "P f  s  phosphorylated  in vitro;  F,  FSV  pi gag-fps isolated from tsFSV-transformed CEFs labelled with [ S] methionine; G, Pr76 35 40  35  isolated cells.  from  the  gag  same  [ S]methionine-labelled  The numbering of methionine-containing peptides  i s according to Pawson et a l . (1981) and is differentiated by a superscript  "S".  through 5 are fps specific. s  Tryptic peptides  I  s  - 75 -  •  - 76 -  3a through 3c and 4) and serine (peptide 5) phosphorylation are contained within the fps region. cleavage  fragments  labelled p l 4 0  g a g - f  P  used  in  To verify the identity of the pl5 these  experiments,  [ S]methionine-labelled 35  P140  g a g _ f p s  ,  and  35  isolated from FSV-infected cells was cleaved  s  with pl5 and coelectrophoresed with The  [ S]methionine-  FAV  VrlQ^  peptide analysis (Figure 3.3).  33K  the  P-labelled fragments.  pl5  were  fragment,  then  intact  subjected  to  FSV  tryptic  This confirmed that the 33K fragment  contains only gag-encoded sequences. To localize phosphorylation sites in C-120K(pl5) more accurately within the fps-encoded region of P l 4 0  g a g - f p s  partial proteo-  l y t i c cleavage fragments were generated with Staphylococcus aureus V8 protease (Houmard and Drapeau, 1972) using the Cleveland gel technique (Cleveland, P140 ~^ gag  ps  et a l . , with  1977).  Digestion  low concentrations  of  of  V8 protease  32  P-labelled yields  two  major cleavage products with apparent molecular weights of 78K and 61K [78K(V8) and 61K(V8)] (Figure 3.4). [ S]methionine-labelled 35  pi40  g a g _ f  Limited V8 protease digestion of P  s  also  produces  these  two  major cleavage fragments (Figure 3.5). Tryptic peptide analysis of the OK  V8 P140  protease gag-fps  (  digestion F i g u r e  3. ) 6  products  of  [ Sjmethionine-labelled  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 i s no apparent overlap  - 77 -  Figure 3.4:  Cleavage  of  Lanes: 32  A through  C,  during  an  with P i  FSV  pi40 g~ P ga  f  with  s  tsFSV  pi40  immune  V8 g a g - f p s  complex  protease. labelled kinase  reaction, purified by SDS-polyacrylamide gel electrophoresis, 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 FSV 35  pi40  of g a g - f  S-labelled  r°S]methionine P  with  s  proteins  V8 were  or  JZ  P-labelled  protease. isolated  3 2  P-  or  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. 1, tsFSV PMO^S^P* in vitro; p gag-fps 140  Lane  labelled  3  2,  obtained  with  [ S]methionine-labelled 35  by  cell-free  Lane 3 2  P  tsFSV  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 this deduction, I phosphorylated  region (Figure 3.6 A and D). To test  8 1  isolated the O120K(pl5) fragment of in vitro-  pi4oS g~ P a  f  s  and  digested  it  with  V8  protease  (Figure 3.7); this generated the same 61K(V8) fragment contained within the intact P l 4 0 expected  if  p gag-fps 140  gag  ~P , f  78K(V8) a n d  but no 78K(V8) fragment.  s  corresponded  6 1 K ( V 8 )  t o  i t s  to  the  This would be  N-terminal  half  of  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 i s resistant to limited V8  protease digestion.  Tryptic phosphopeptide analysis of the N-terminal 78K and C-terminal 61K V8 protease fragment [N-78K(V8) P140  gag-fps  f  r o m  32  P-labelled  and C-61K(V8)] of  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 Pl40 ~^P gag  portion s  of  pi40  g a g - f  P . s  A  similar  analysis  of  phosphorylated in vitro in the immune complex kinase  - 82 Figure 3.6:  Tryptic peptide analysis of V8 protease cleavage fragments  of  FSV  pi40  g a g  ~P . f  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: of  A, N-78K(V8) produced by V8 cleavage  [ S]methionine-labelled 35  tsFSV  pi4o8ag-fps  obtained by cell-free translation of FSV(FAV) polyadenylic 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 P140  P  gag_f  phosphorylated  s  in vivo  (18 hour  label-  ling); C, N-78K(V8) obtained by V8 protease cleavage of tsFSV  pi40  C-61K(V8)  g a g - f  P  phosphorylated  s  produced  by  V8  in vitro;  protease  [ S]methionine-labelled  D,  cleavage  tsFSV P i 4 0  35  of g a g _ f  P  s  obtained by cell-free translation of FSV(FAV) polyadenylic 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 ~^P gag  s  phosphorylated  in vivo  (18-hour  label-  ling); F, C-61K(V8) obtained by V8 protease cleavage of tsFSV  pi40  g a g _ f  P  s  phosphorylated  in vitro.  The  numbering of methionine-containing peptides i s according to Pawson, et a l . (1981) and i s differentiated by a superscript "S". specific.  Tryptic pepties I  s  and 5  s  are fps  - 84 -  Figure 3.7:  Mapping P  V8  140gag-fps  protease  #  cleavage  T h e  fragments  of  pi e g- P a  t s F S V  f  FSV was  s  4 0  32 labelled  with  P^  during  an  immune  complex  kinase reaction and cleaved with NP40-disrupted virions. Uncleaved  pi40g g~ P a  f  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-polyacrylamide  gel.  Lanes:  A,  tsFSV  PMOS - "* * ;  120K(pl5) fragment; C, 33K(pl5) fragment.  3  6  1  3  B,  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 observation that  the N-78K(V8)  from in vitro-phosphorylated  i s more heavily labelled with corresponding  fragment  (data not shown).  It  from  3 2  Pl^^S *? -  8  P relative to C-61K(V8) than the in vivo-phosphorylated  s  pi4QB&&~fP  i s possible that the in vitro-phosphorylated  peptide 2 i s near the site of pl5 cleavage, since i t i s not easily recovered in the pl5 cleavage fragments. The results of these mapping experiments are shown diagramatically 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 PMO - ""-^ with a thermolabile protein kinase a c t i 63  8  8  vity (Pawson, et a l . , 1980).  To determine whether this temperature  sensitivity reflects any change in the amino acids sequence immediately surrounding  pi^S  3,6  "-^  8  phosphorylation  sites,  I  analysed  tryptic phosphopeptides of i t s temperature-resistance derivative, FSV (Lee, et a l . , 1981). PMO^S ^ , -  labelled  8  with  isolated 32  the tr  Figure 3.9 shows that t r FSV and ts FSV from  FSV-transformed  P-orthophosphate,  have  CEFs  tryptic  metabolically phosphopeptides  - 87 -  Figure 3.8:  Cleavage P140  sites  g a g - f p s  text. tryptic  for  pl5  and V8  protease  on FSV  yielding the fragments described in the  The numbers indicate the putative location of phosphopeptides  discussed in the text.  within  FSV  pi40  g a g - f  P  s  as  78 k-  V8-  61k  P15 33k 00 00  1  NH, "  gag— p19, p10, *p27  120k-4 4,5,  2?  fps  3a-3c  COOH  - 89 -  Figure 3.9:  Tryptic P140  phosphopeptide  gag-fps  FSV  >  p  130  f  ga  FSV  8  gag-fpS  )  o  r  of  trFSV  and  PRCII  PWOS^-fP ,  FSV  pi3o g- P  P105 ag- P . g  analysis  P R C I I  pl05  f  8  8  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  [)(- P]ATP  anti-pl9 P140  after  32  immunoprecipitation  serum from transformed  gag-fps  )  pl  3 gag-fp 0  S)  CEFs.  with  Gel-purified  o r  pl05  gag-fps  was then digested with trypsin and separated in two dimensions on thin- layer cellulose plates. Tryptic digests  were  phosphorylated phorylated  as follows: in vivo;  in vivo;  B,  A,  trFSV  FSV P 1 3 0 23  D, PRCII P105 ~ P  in vitro;  PRCII  in vivo.  gag  C, trFSV P M O - " ^  lated in vitro; E,  pi40  P105  gag  f  "  f p 8  g a g  8  6  8  ~  g a g - f  fp8  P  8  phos-  phosphory-  phosphorylated phosphorylated  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 ~ . The additional spots panels B and E have not been analysed further. gag  fps  in  - 91 identical to those of their p i 4 0  proteins phosphorylated in  g a g - f p s  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. this variant is phosphorylated in vivo with  The P I S O 3 2  83,6-  ^  8  from  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 serine phosphorylation site, or that the sites are completely different.  PRC! I is an independent isolate of a transforming virus containing 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  P130  (Huang,  g a g - f p s  et a l . ,  1984).  PRCII  P105  gag  ~  fps  phosphorylated in the immune complex kinase reaction has a tryptic phosphopeptide map similar to that of in vitro-phosphorylated tsFSV L5 and  trFSV  P140  g a g - f p s  ,  suggesting  that  it  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 i s 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 i s  contained within a region of FSV p i 4 0 PRCII P 1 0 5  g a g - f p s  .  g a g - f p s  that is deleted in  - 92 -  3.3 Discussion Phosphoamino acid analysis of  FSV p i 4 0  indicates  g a g _ f p s  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 i s highly conserved between the transforming proteins of different FSV variants. N-terminal gag region of p i 4 0 sites shared with Pr76  gag  The  contains minor phosphorylated  g a g - f p s  (Pawson, et a l . , 1981 and this study),  but surprisingly i t is also phosphorylated at a tyrosine residue contained within an acidic tryptic phosphopeptide.  The importance of this  gag phosphotyrosine site to the functional activity of is unknown.  pi40  g a g - f p s  Clearly, the tyrosine phosphorylation within the gag i s  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). P140  g a g - : fp s  The major ,  including  fps-specific two  phosphorylation  phosphotyrosine  sites  residues  of and  FSV 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 fragments  with  kinase  activity  support  the  suggestion  trypsin that  - 93 the C-terminal sequences of the ASV transforming proteins are highly conserved because they encode the kinase domain. sequence  surrounding  the  pi40 ~ P gag  f  The conservation of  phosphotyrosine  s  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 S P ga  -f  itself and  s  i t s kinase activity.  Several groups have compared the phosphorylation sites from a number of  different  sequencing  (Neil,  viral  et a l . ,  transforming proteins by using micro1981; 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  P105  gag  ~  fps  and  FSV  pi40  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 i n i t i a 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 t s side chain amide group during the  - 94 -  tryptic mapping procedure may yield a tryptic peptide having pyroglutamic acid at i t s N-terminal end (3b) which i s a less positively charged species at pH2.1 compared to the unmodified peptide containing glutamine at i t s 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 ~*P gag  s  i s 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 reaction, perhaps this site of tyrosine phosphorylation i s not the product of  pi40 g~^P ga  s  autophosphorylating  activity  phorylated by a cellular tyrosine kinase.  but  rather  is  phos-  Alternatively, the lack of  phosphorylation within peptide 4 could reflect the a r t i f i c i a l nature of the in vitro immune complex kinase assay which would also account for the  phosphorylation  P140 ~"^P gag  s  of  peptide  phosphorylated in vitro.  2  which  is  only  seen  with  This lack of phosphorylation  within peptide 4 does not affect the apparent kinase activity of P140 ~^P gag  s  as measured in vitro.  It  appears doubtful  peptide 4 is present in PRCII P 1 0 5 ~ P . gag  f  s  However, i f  whether it  is  absent from this transforming protein i t clearly does not affect i t s 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 i s involved  in the phosphorylation of P l 4 0  in FSV transformed cells.  g a g _ f p s  tyrosine  residues  Experiments described in the following  chapters were designed to investigate the relationship between the phosphorylation of the FSV transforming protein and i t s enzymatic and biological activities.  CHAPTER 4  4.0 Oligonucleotide-directed Mutagenesis of Fujinami Sarcoma Virus: Evidence that Tyrosine Phosphorylation  of p gag-fps 130  Modulates i t s Enzymatic and Biological Activities. 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 i s 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 c e l 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, phosphoglycerate mutase and lactate dehydrogenase) 1981b; Cooper, et a l . , 1983a).  FSV P i 4 0  gag  ~P f  (Cooper and Hunter, s  and i t s 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 ~ fP gag  concentrations.  :  s  that are within physiological  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 phosphorylation in the induction of c e l 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 t s 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 serinespecific protein kinases (Cohen, 1982).  However, such a function has  yet to be demonstrated for any case of reversible tyrosine phosphorylation;  indeed substitution of RSV p 6 0  src  lanine destroys the major site of p 6 0  tyrosine-416 with phenylasrc  tyrosine phosphorylation  but has no obvious effect on src transforming ability or kinase a c t i vity of p 6 0  (Snyder, et a l . , 1983).  src  showed that P 1 3 0  gag_f  P  s  In the previous chapter I  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  investigate  5 1  as a model substrate to  whether tyrosine phosphorylation can in fact modify protein function. The  major  site  of  Piso^g-fP  phosphorylation  8  in vivo,  as  in vitro, i s tyrosine-1073 which i s located in the C-terminal kinase domain and i s homologous to the major site of tyrosine phosphorylation in  RSV  p60  src  and  Y73  Shibuya and Hanafusa, 1982).  P 9 0 ^ ^  e  s  (Kitamura,  et a l . ,  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 lanine.  g a g _ f p s  to phenyla-  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 phenylalanine.  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  gag_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 i s 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 viral inserts were  propagated in E. coli JMlOl, and those phage ssDNAs with the FSV strand complementary to the oligonucleotide were identified by their ability 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. coli 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 ~ P gag  f  s  mutated to TTT  using the  cleotide displayed in Figure 4.1(b). phage ssDNAs were dotted  onto  oligonu-  Putative mutant  nitrocellulose  and  screened for their abilities to hybridize with the 32 P-labelled  mutagenic oligonucleotide  tions of increasing temperature.  under  condi-  - 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 I N 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 i s 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 differences  between  the pl30  gag-fps proteins  encoded  by  wtFSV  and  FSV-F(1073) resulted from the conversion of tyrosine-1073 to phenylalanine 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 phenylalanine1073 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) i s identical to wtFSV in the region of interest (Figure 4.3). Any biological activity FSV-Y(1073)  of wtFSV that i s altered in the mutant  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 dideoxynucleotide chain termination method.  The codons for amino  acid 1073 are arrowed in each case.  wtFSV WILD TYPE  FSV-F(1073) FSV-Y(1073) 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 abilities to transform rat-2 cells.  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 10 cells, and by three weeks 6  approximately five times more foci were visible, though many of these may have arisen by secondary spread of transformed cells.  In con-  trast, rat-2 cells transfected with FSV-F(1073) mutant DNA showed no evident morphological change until 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 cells,  - 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). lines  of  wtFSV-transformed  rat-2  cells  (E)  Cell 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).  8  FSV  FSV  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).  All  such morphologically transformed cell 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 c e l 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 triplicate.  Approximately  three weeks post-injection tumors appeared at the site of injection in rats that had been injected with wtFSV or revertant transformed cells. 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 cells,  but this occurred only after 5  months following injection and probably reflects the properties of this immortalized, continuous c e l l line.  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.  - Ill -  Tumors from these animals were excised, grown in tissue culture and subsequently assayed for the expression and activity of P 1 3 0 as described below. cells  g a g - f p s  Tumors induced by mutant FSV-F(1073) transformed  contained cells  which expressed  P130  characteristic  g a g - f p s  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 Wildtype and Mutant P 1 3 0  gag  ~  fps  Proteins  Equal numbers of cells transformed with wtFSV, FSV-F(1073) or with FSV-Y(1073) were labelled with [ S]methionine for 16 hours, 35  lysed and immunoprecipitated with a n t i - p l 9 amounts of labelled P 1 3 0  g a g _ f p s  Pulse-chase experiments have  the wtFSV and FSV-F(1073)  approximately  the  same  Equivalent  were immunoprecipitated from each  line of transformed cells (Figure 4.5). shown that  serum.  gag  turnover  P130  rates  proteins  g a g - f p s  (data  not  have  shown)  and  o r  [  S]methionine-labelling  measure of P 1 3 0  gag  ~  fps  can levels.  therefore  be  used  as  a  relative  The mobilities of the wtFSV and  revertant proteins were identical, demonstrating that the mutagenesis procedures had not grossly affected the FSV coding sequence. PISO ^ ^ 53  -  8  Mutant  encoded by FSV-F(1073) migrated slightly more rapidly  than the wild-type or revertant proteins, possibly as a result of decreased phosphorylation. or  revertant  P130 ~^ gag  ps  In a parallel experiment wild-type, mutant proteins  were  immunoprecipitated  from  - 112 -  Figure 4.5  Analysis of wild-type, mutant and revertant FSV-transformed  rat-2  cells  for  FSV  P130  gag  ~  fps  synthesis  and tyrosine-specific protein kinase activity.  Normal  and transformed rat-2 cells were labelled for 16 hours with  100  uCi  with a n t i - p l 9 phoresis  to  gag  [ S]methionine,  immunoprecipitated  serum and examined by gel electro-  identify  pi30  g a g _ f  P  s  (lanes  A-E).  Cells from duplicate unlabelled cultures were immunoprecipitated in identical fashion and then incubated with 5 ug of acid-denatured enolase in the presence of 10 mM MnCl  2  and 2.5 uCi  30°C (lanes F-J).  ["^- P]ATP 32  for  15 minutes  at  Samples were anlaysed by electro-  phoresis through a 7.5% SDS-polyacrylamide gel and autoradiography.  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 u n l a b e l l e d c e l l s and the immune complexes incubated  qp with  MnCl ,  [ J - P]ATP  2  enolase  identical  soluble,  rabbit 6  I n t h i s r e a c t i o n wtFSV P I S O - " ^  and a l s o phosphorylates  t o that phosphorylated  f i b r o b l a s t s (Cooper,  denatured 83  (Figure 4.5).  phosphorylated  and  et a l . ,  enolase  8  muscle i s auto-  at a single tyrosine  i n FSV-transformed  chicken  embryo 1  1984a).  Mutant FSV-F(1073) PISO^S"* *  3  c l e a r l y f u n c t i o n s i n v i t r o as a p r o t e i n kinase but i s apparently l e s s active  in  phosphorylating  enolase  than  wtFSV  or  FSV-Y(1073)  p gag-fps. 130  was  The extent of enolase phosphorylation i n each case normalized t o the amount of immunoprecipated P 1 3 0 ~ ^ P by gag  8  qp determining 4.1).  the  P  or  S  cpm  i n the relevant  The mutant p r o t e i n i s approximately  bands  (Table  f i v e - f o l d less active i n  enolase phosphorylation than the wild-type o r revertant p r o t e i n s when assayed i n t h i s way. The considerable decrease p l 3 0  i n r a d i o l a b e l l i n g of FSV-F(1073)  gag-fps itself  i n the immune complex kinase  r e a c t i o n (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 y r o s i n e autophosphorylation had been destroyed by s u b s t i t u t i n g t y r o s i n e 1073 w i t h phenylalanine. lated  C-terminal fragments of i n vitro-phosphory-  mutant p l 3 0  gag-fps generated  by  cleavage  with  V8  protease  were poorly phosphorylated compared with corresponding fragments of the wtFSV p r o t e i n (data not shown). To confirm that the expected amino a c i d s u b s t i t u t i o n s had been introduced a t residue 1073 and t o define the e f f e c t of these s u b s t i t u t i o n s on phosphorylation of the mutant and  - 115 -  TABLE 4.1 Quantitation of the Kinase Activities of P130 "" P gag  f  s  Proteins Encoded by Wild-Type, Mutant and Revertant FSV DNAs Relative Phosphorylation - in vitro 8  by P l 3 0 Source of PISOgag-fPS  Enolase  wtFSV FSV-F(1073) FSV-Y(1073)  7.5 1.4 6.0  a  gag  ~P f  s  pisoga-g-fps (autophosphorylation)  9.7° 2.2 ND  b  0.6 0.1 0.6  d  Immune complex kinase reactions were performed as shown in Figure 4.5  following immunoprecipitation of  P130  from rat-2  g a g - f p s  cells transformed by wtFSV, FSV-F(1073) or FSY-Y(1073). extent  of  enolase phosphorylation  or  P130  gag-f  P  s  The  autophos-  phorylation was measured by counting appropriate dried gel slices for  3 2  P.  The  amount  of  P130  gag_f  P  s  in  each immunopre-  cipitate was estimated by labelling duplicate dishes of cells with [ S]methionine,  immune-precipitating  35  counting the gel-purified protein. ratios  of  P cpm incorporated  PISO^^^P  The values shown are the into  the substrate  cpm in P130 ~ P . gag  f  s  b,c Values are from two separate experiments. ND, not done. d  Mean of two separate experiments.  and  8  to  S  - 116 -  revertant proteins tryptic digests ofwtFSV, FSV-F(1073) and FSV-Y(1073) pl  3 gag-fps 0  or  j  had  w n i c n  [ S]methionine  been or  radiolabelled  in vivo  autophosphorylated  with  3 2  in vitro  P  i  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 migrating peptide species.  A tryptic phosphopeptide map of P l 3 0  gag  ~^  ps  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 contain tyrosine-1073 whereas the remaining two correspond to less prominent sites of phosphorylation (Chapter 3).  The tryptic peptide encom-  passing tyrosine-1073 also possesses a methionine residue, and a tryptic  peptide  map  of  [ S]methionine-labelled  wtFSV pisoS - "* 8  35  6  158  should therefore contain radiolabelled peptides corresponding both to the phosphorylated species 3a-3c and their non-phosphorylated counterparts in a ratio dependent on the stoichiometry of phosphorylation. A  map of  mutant  FSV-F(1073)  PISOS - ""* 3 6  158  labelled  by  in vitro phosphorylation (Figure 4.6, map F) i s entirely lacking spots 3a-3c but retains the two minor tryptic phosphopeptides, confirming that 3a-3c represents phosphorylation of tyrosine-1073. the tryptic phosphopeptide map of  In contrast,  revertant p gag-fps 130  phosphorylated  in  an  immune complex  is  identical  to  that  of  wtFSV  - 117 Figure 4.6  Tryptic peptide analysis of P130  gag-fps encoded by  wild-type,  mutant and revertant  FSVs. pl30  gag-fps  isolated from transformed rat-2 cells labelled in vivo with  [ S]methionine  (A, D, G)  35  orthophosphate  (B, E, H)  or  or  with  labelled  3 2  with  P-  3 2  P  in vitro by autophosphorylation in immune complex kinase reactions tryptic  (C, F, I)  was subjected  to 2-dimensional  peptide mapping. pl30  gag-fps was  isolated  from rat-2 cells transformed with wtFSV (A, B, C) or with  FSV-F(1073)  (G, H, I).  (D, E, F)  tsFSV  tsFSV(FAV)-infected [  S]methionine  37°C' (J) labelled  or  P140 CEFs  (J, K). at  proteins  or  FSV-Y(1073)  s  was  isolated  from  which  were  labelled  with  gag_f  P  with  CEFs  were  41.5°C (K). 32 and  maintained  at  [ S]methionine35  P-labelled  proteins  were  oxidized, digested with trypsin and separated in two dimensions in identical fashion. with an 'VD".  Origins are marked  Electrophoresis at pH 2.1 i s 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) (see Chapter 3). or Chapter 3. The double spot to the right of phosphopeptide 4 in maps E and F and the spot above 3c in map F are variable and have also been observed in wtFSV Pl gag-fps 30  - 118 -  35  w I  S MET A  WTFSV  00 P IN VIVO  WTFSV  30 B  P IN VITRO  WTFSV  L D T Y P E  - 3c 3b  y  3c  v  1 »  3b" p  3b -  ^ #  FSV F(1073)  D FSV~F(1073)  £  FSV F(1073)  H  FSV-Y(1073)  M  u  • 3 c  T A N T  R E V E R T A N T  3b  .  •  FSV-Y(1073)  G  FSV Y(1073)  *  3b^  .  *^3b  T E M P E R A T U R E  tsFSV  37°C  S E N S I T I V E  e  p  4 •  3a  p  Ji.D-V  %  p  3b tsFSV 41.5°C  K  3c' 3b  • • * 3c  3b  3b*  ¥  p y  p y  3a  I  - 119 -  pl30  gag-fps  (  F i g u r e  4  .  ^  6 )  j.).  findings  These  shew  tyrosine-1073 i s indeed the major site of wtFSV piSO^S^P  that auto-  8  phosphorylation, and that this phosphorylation site i s lost in the mutant protein but restored in revertant P 1 3 0  gag  ~ .  To under-  fps  take a more detailed structural analysis of the  pro-  teins, and to identify the mutant phenylalanine-1073-containing tryptic peptide pl30  I  used  in vivo  labelling  with  [ S]methionine.  wtFSV  35  gag-fps contains  [ S]methionine-labelled 35  tryptic  peptide  species which co-migrate with tryptic phosphopeptides 3a, 3b and 3c of 32  P-labelled  PISOS " "^ 3  6  (Figure  8  4.6,  Map  A).  Presumably  they correspond to the phosphorylated form of the tyrosine-1073containing tryptic peptide with an unmodified (3c) or modified (3a, 3b) N-terminal 3cP~  glutamine,  and  are  3aP~ ,  designated  v  to indicate the presence of a phosphotyrosine.  v  3bP~  v  and  To confirm  the identity of these methionine-containing tryptic peptides I have used a variant of FSV which i s temperature-sensitive for transformation (tsFSV L-5)  and which encodes a P 1 4 0  gag-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  grown at 3aP~ -3 P~ v  C  gag  14Q  f  37 °C contains in  v  f r o m  s  from infected chicken embryo fibroblasts [ S]methionine-labelled tryptic  similar  (Figure 4.6, map J), p me-fps  ~P  yield  to  wtFSV  peptides P130^^~fPs  but these peptides are barely detectable in  ^fected cells maintained at 41.5°C (Figure 4.6,  - 120 -  map K), as would be expected i f they contained a reversibly phosphorylated tyrosine residue.  I then analysed a tryptic digest of methionine-  labelled mutant FSV-F(1073) could  detect  no  spots  P130  (Figure 4.6, map D) and  g a g _ f p s  corresponding  to  phosphorylated  3a  p - y  -  3 p-y, consistent with predicted substitution of phenylalanine for C  tyrosine-1073. mutant P 1 3 0 nated 3b ,  In  gag  3c  y  ~ y  fps  addition  to  the  lacks two further  absence  of  p  3aP~y-3c ~'y,  wild type peptides  on maps of wild-type  and revertant  (desig-  P130  but has acquired two novel peptides (designated 3b and 3 c ) . f  3b  y  and 3c  P130  g a g - f p s  y  f  g a g - f p s  )  The  wild-type peptides missing from the digest of mutant migrate as i f  more hydrophobic  they were less negatively charged and  than the phosphorylated  Sb " 15-  57  and 30^^.  My  interpretation of these data is that wild-type peptides 3b and 3c y  y  correspond to the non-phosphorylated forms of the tyrosine-1073-conf  taming tryptic peptide and that the mutant peptides 3b  f  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 , but move more rapidly in the chromatographic dimension of y  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  identical to those of wtFSV P 1 3 0 of  [ S]methionine-labelled 35  gag  ~ . fps  revertant  gag  ~  fps  are  The tryptic peptide map 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 i s the major site of P l 3 0  gag  ""  tyrosine phosphorylation, that this  fps  residue has been substituted with phenylalanine and i s no longer phosphorylated 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^^~ P f  s  is  residues in FSV-transformed cells.  phosphorylated  at  three  tyrosine  A minor and variable site Of tyro-  sine phosphorylation lies 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). residues are gag-fps  also  phosphorylated.  wtFSV  Several serine and  FSV-F(1073)  P130  proteins  were  isolated  from  32  P-labelled  trans-  formed rat-2 cells and analysed for phosphoamino acid content and by tryptic phosphopeptide mapping. The mutant protein contained approximately two-fold less phosphotyrosine, relative to i t s total phosphoamino acids, than the wild-type protein (Figure 4.7, Table 4.2) and this i s explained by the absence of tryptic phosphopeptides 3a-3c from digests map E).  of  in vivo-labelled  FSV-F(1073)  P130  gag  ~  fps  (Figure 4.6,  The mutant protein i s 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  PISO^^P .  Rat-2  8  cells transformed with wtFSV (A) or mutant FSV-F(1073) qp  (B)  were  hours,  labelled  lysed,  with  P-orthophosphate  immunoprecipitated  with  for  4  anti-pl9  gag  serum and the immunoprecipitates analysed by gel electrophoresis.  32  P-labelled  pi30  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 autoradiography.  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 P130  gag-fps  and Total Cellular Protein PISO^^PS Phosphoamino acid  Total Cell Protein a  wtFSV  FSV-F(1073)  rat-2  wtFSV  p-serine  65°  82  95  90  91  p-threonine  13  8  5  9.7  8  p-tyrosine  22  10  .07  .39  .27  Radioactivity isolated  from  in 32  individual P-labelled  phosphoamino cells  or  of  acids total  FSV-F(1073)  of  b  P130 ~-*-P  cellular  gag  3 2  s  P-  labelled protein was determined by scintillation counting of aspirated thin-layer cellulose spots following electrophoretic separation of acid hydrolysates (Figures 4.7 and 4.8).  a  wtFSV-transformed rat-2 cells.  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 i s not apparent in the map shown in Figure 4.6. phosphorylated pl30  at  In addition FSV-F(1073) P 1 3 0  the  same  judged  by  serine  sites  in vivo  g a g - f  P  as  s  is  wtFSV  gag-fps as  containing tryptic peptides.  the  migration  of  phosphoserine-  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 of 32  revertant  FSV-Y(1073)  g a g - f p s  .  A tryptic phosphopeptide map  PISO^^P*  5  labelled  in vivo  P i (Figure 4.6, map H) once more contained peptides 3a-3c.  with In  a series of independent tryptic digests the only consistent difference between  the  tryptic  FSV-Y(1073) P l 3 0  gag  phosphopeptides  ~P f  s  of  wtFSV,  FSV-F(1073)  and  was the absence of 3a-3c from the mutant  protein. Transformation by FSV induces an increase in total cell phosphotyrosine 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  *P-orthophosphate  J  and  analysed for whole cell phosphoamino acid content (Figure 4.8; Table 4.2). In one experiment wtFSV-transformed cells had 5.6-fold more phosphotyrosine than normal rat-2 cells, 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 cell 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 Pi  and  total  acid-hydrolyzed  cellular  protein  was  extracted,  and separated in two dimensions by  electrophoresis at pH 1.9 and pH 3.5. The mobilities of marker phosphoamino acids are phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) as identifed by ninhydrin staining.  - 127 -  40  - 128 -  4.3 Discussion The substitution of phenylalanine for tyrosine at residue 1073 of  does not abolish the ability of FSV to trans-  ^ 1 3 0 ^ ^ ^  form rat-2 cells.  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) i s rapidly transforming, and this suggests that the mutant protein i s 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, i s reduced five-fold compared with wtFSV or revertant reduced  PlSOgag ^ . -  kinase  8  activity  It of  is  reasonable to  the  mutant  propose  FSV-F(1073)  that  the  pi30  g a g _ f p s  results from i t s inability to become phosphorylated at residue 1073 and that this reduced enzymatic activity i s responsible for the i n e f f i ciency of the mutant FSV-F(1073) in inducing transformation of rat-2 cells.  This conclusion rests on the assumption that phenylalanine at  position 1073 does not disturb the conformation and activity P130  g a g - f p s  of  .  The functional differences between the mutant and wild-type P130  gag-fps can be attributed to the substitution at residue 1073,  since the wtFSV and revertant  FSV-Y(1073)  DNAs and  P130  gag  ~  fps  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 i s 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). purified insulin receptor or RSV p 6 0  src  Preincubation of  under conditions promoting  their autophosphorylation enhances their subsequent ability to phosphorylate 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 a c t i vity but is not detectably phosphorylated at tyrosine in cultured cells (Mathey-Prevot, et a l . , 1982). of P 1 3 0 ~ ^ gag  ps  It is possible that the phosphorylation  in vivo represents a unique feature of the v i r a l  transforming protein which fortuitously leads to increased kinase a c t i vity.  Perhaps, NCP98 becomes transiently phosphorylated at tyrosine in  the animal in response to a specific environmental signal (which i s 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 P130  phosphorylation  g a g _ f p s  at  tyrosine-1073  results  in  constitutive enzymatic activation which in turn i s important in the induction of unregulated cellular proliferation. The FSV-F(1073)  basis is  for  the  unknown.  long  latency  of  transformation  Recombination with c-fps  resulting  by in  restoration of the codon for tyrosine-1073 does not seem likely since a l l of the P I S O  836-  ^  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 gel electrophoresis.  g a g - f p s  in SDS-polyacrylamide  It i s possible that the latent period reflects  the requirement for activation of a cellular gene that co-operates with mutant  PISO " "^ 69  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  tyrosinespecific protein kinase (Whitlock, et a l . , 1983).  is  also  a  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 cells.  - 131 -  The phenotypic consequences of changing tyrosine-1073 of FSV P130  are  g a g - f p s  much more  dramatic  corresponding mutants of RSV src. tyrosine  phosphorylation,  plSOgag-fps  than  those  for  The major site of RSV p 6 0  tyrosine-416, which  tyrosine-1073  observed  has  recently  is  src  homologous  with  changed  to  been  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 RSV src.  for the pSR-XDT10-l deletion mutant of  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  p60  that  the  substitution  of  src  tyrosine-416  with  phenylalanine has no apparent effect on transformation of mouse fibroblasts  in  culture  or  p60  src  contrast to my results with FSV.  kinase activity  in vitro,  in  Although further investigations  reveal that this src mutant i s 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 a c t i vity  of  FSV PISO^ ^ ^ 3  -  81  other  than tyrosine  involved in i t s transforming ability.  phosphorylation  is  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 activity  is  consistent with the general concept that  g a g _ f p s  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 t s oncogenic action.  - 133 CHAPTER 5  5.0  The Protein Kinase Activity of FSV PISOS - "^ 3  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 i s of interest as i t likely 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 substrate 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 tyrosine residue (Gallis, et a l . , 1984a).  et a l . , 1983; Guild, et a l . , 1983; Cooper,  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 i s probably also an important recognition factor.  In any case, the studies using  synthetic peptides as substrates suggest that tyrosine kinases have a strict specificity for phosphorylating tyrosine residues (Pike, et a l . , 1982; Hunter, 1982; Wong and Goldberg, 1983b).  In contrast, there i s 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 product 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 lipid 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 cells, which allows the specificity and dynamics of phosphorylaton to be examined in vivo.  As a consequence, such studies w i 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 previously described (section 4.2.1). The oligonucleotides used to change the TAT codon for tyrosine-1073 within P 1 3 0  gag  ~  fps  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 i s 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 P130 . 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 P and their encoding nucleotide sequence. g a g - f p s  gag-f  s  .... ArgGlnGluGluAspGlyValTyrAlaSerThrGlyGlyMetLys  .... C  .... CGGCAGGAGGAGGATGGTGTCTATGCCTCCACGGGGGGCATGAAG .... 3' 5* - TGGTGTCTCTGCCTCC - 3'  Ser FSV-S(1073)  ACT  Thr FSV-T(1073)  GGT  Gly FSV-G(1073)  TAT  Tyr FSV-Y(1073)  WILD TYPE FSV  - 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-1073  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 immunocompetent rats, but did so with a longer latent period than wt or revertant transformed cells.  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. were similar in biological  activity  to  FSV-F(1073) mutant described in Chapter 4.  the  The mutants  previously  isolated  - 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 cells; 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 phorylation and associated kinase activity.  g a g - f p s  expression, phos-  To identify P l S O ^ g - ^ -  8  qc  cells  were  metabolical ly  labelled  with  [ S] methionine  and  lysates were immunoprecipitated with a monoclonal antibody directed against  avian  The  s  pl9^ .  amount  porated reflects the level of P 1 3 0 among the different  of  g a g - f p s  [ S] methionine 35  incor-  expression which varied  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 levels.  into  P130 S~* , ga  ps  rather  than  decreased  expression  First, a clone of transformed cells isolated following trans-  fection with revertant FSV-Y(1073) DNA had approximately 5-fold less gag-fps  pl30  than a clone of  mutant transformed cells,  yet  the  latter cells were not as overtly transformed as the revertant transformed 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 pl30  gag-fps in  trans-  formed rat-2 cells and quantitation of their respective tyrosine protein kinase activities.  4a: Normal or  transformed rat-2 cells were labelled for 16 hours with OCT  100  ucl  anti-pl9 phoresis  [ S]methionine, serum  gag  to  and  immunoprecipitated  examined  by  gel  with  electro-  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 identical fashion and then incubated with 5 ug of aciddenatured enolase in the presence of lOmM MnCl  2  2.5  30°C.  uci  [tf- P]ATP 32  for  15  minutes  at  and  Samples were analysed by electrophoresis through a 7.5% SDS-polyacrylamide gel and autoradiography.  Lane F,  rat-2 cells; 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 PISO ^""^ 63  proteins,  8  enolase was included in  the immune complex kinase reactions as an exogenous substrate for tyrosine phosphorylation (Figure 5.4b).  Quantitation of the extent of  enolase  and wt  phosphorylation  by  mutant  29  -  PISO ^ "^  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 deterqp  mining the ratio of °^P-enolase, reaction  to  that  of  the  labelled by an in vitro kinase  respective  PISO ^ "^  labelled with [ S]methionine (Figure 5.4). 35  23  -  metabolically  8  This ratio represents  the extent of kinase activity per amount of PISC^ ^"""^ present in 9  8  the reaction, and the values reported are an average of four independent experiments. Cell lines that were cloned from each of the revertant FSV-Y(1073)  transformed cells were shown to  express  P130  g a g - f p s  with similar in vitro kinase activities and electrophoretic mobilities as  wtFSV  PISO  636-  ^  8  (data  not  biochemical data indicated that  shown).  Both  the PlSOgas-^PS  biological  and  proteins encoded  by the revertant DNAs were structurally and functionally identical to  - 147 wtFSV  P130 "" gag  and  fps  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) P130 ~^ gag  ps  PISO ^"^ 6  8  and  the  threonine-1073  of  FSV-T(1073)  do not become phosphorylated either in the rat-2 cells  expressing these proteins or in vitro during immune complex kinase reactions. slightly  The electrophoretic mobilities of the mutant proteins were greater  than those of  the wild-type  PISO  may result from decreased phosphorylation (Figure 5.4).  68-8  "^ , ps  which  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),  the major autophosphorylation site.  consistent with the loss of  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. P130  g a g - f p s  Phosphorylated  peptides  3a-3c  of  wtFSV  are derived from a single tryptic phosphopeptide con-  taining residue 1073 (Chapters 3 and 4) and were clearly absent in tryptic  peptide  phosphorylated P130  g a g - f p s  maps of in vitro  FSV-S(1073) (Figure  immunoprecipitated  or  5.5).  from  cells  FSV-T(1073) Similar  P130 analysis  metabolically  gag  ~  fps  of  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 P130  proteins  g a g _ f p s  labelled  in vitro.  p gag-fps 130  encoded by  wild-type  and mutant  FSVs  were labelled in an immune complex kinase assay following  immunoprecipitation with a n t i - p l 9  transformed cells.  gag  serum from  The wt and mutant  P130  g a g _ f p s  proteins were gel purified, digested with trypsin and separated in two-dimensions on thin-layer plates.  cellulose  Electrophoresis at pH 2.1 was from left to  right with the anode on the l e f t , and chromatography was from bottom to top. Tryptic  digests  An "0" indicates the sample origin. were  as  follows:  a,  wtFSV;  FSV-S(1073); c, FSV-G(1073); d, FSV-T(1073).  b,  The spot  that is not numbered and occurs to the right of 3c i s 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 phosphorylated when substituted P130Bae-*ps  for  a tyrosine  at  residue  1073 within  #  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 substitutions 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  5.2.4  g a g - f p s  .  Tyrosine Phosphorylation of wtFSV and Mutant FSV P 1 3 0  gag  ~  fps  from Transformed Cells  Phosphoamino acid  analysis  of  mutant  Pl30  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 gag-fps pl30  (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  P130  g a g - f p s  proteins.  However, the loss of tyrosine-1073 did not appear to qualitatively  - 151 -  Figure 5.6:  Tryptic  phosphopeptide  encoded  by  pl30  gag-fps  analysis  wild-type  and  labelled  ^ 5  of  mutant with  3 2  P130  gag  FSVs. P  ~  fps  FSV  in vivo  by  incubation of wtFSV transformed cells with 32 P-orthophosphate for 12 hours and mutant FSV oo  transformed hours.  cells  The  32  with  P-orthophosphate  P-labelled  P130  gag_f  P  for  4  proteins  s  were isolated by subsequent immunoprecipitation of the labelled  protein  with  purified PISO^^^P  81  anti-pl9  serum.  Gel-  was then digested with  trypsin  gag  and separated by electrophoresis at pH 2.1 in the f i r s t dimension and chromatography in the second dimension. The anode i s to the left and the cathode to the right. Tryptic  digest  were  as  follows:  A,  wtFSV;  FSV-S(1073); C, FSV-G(1073); and D, FSV-T(1073).  B,  - 152 -  - 153 -  Figure 5.7:  Phosphoamino acid analysis of wtFSV and mutant FSV P130  g a g _ f p s  proteins.  Rat-2 cells transformed with  wtFSV or mutant FSV [FSV-S(1073); FSV-T(1073); (1073)]  were  labelled  with  lysed,  immunoprecipitated  for  P  32  with  ±  12  anti-pl9  FSV-G hours, serum  gag  and the immunoprecipitates separated by gel electrophoresis.  32  P-labelled  P130  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). counts loaded were as follows: FSV-S(1073),  1100 cpm;  FSV-T(1073), 308 cpm.  The number of Cerenkov wtFSV,  FSV-G(1073),  1300 cpm;  540 cpm;  and  - 154 -  - 155 -  affect the phosphorylation of other tyrosine and serine residues within PiSQgag-fps^  The  mutant  Piao  6 8 8 -  ***  proteins  8  were  still  phosphorylated at tryptic phosphopeptide 4, which represents the other major  tyrosine  in vivo.  site  in  wtFSV  P130  g a g - f p s  phosphorylated  only  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) Figure 5.6.  P130  gag  ~  fps  shown in  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  between  the  PISO ^ ^ 63  12 hours,  -  8  tryptic proteins  with  P  i f  the  only  phosphopeptides  of  was the absence of  consistent wtFSV 3a-3c  and  difference mutated  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 chapter.  g a g - f p s  described in the preceding  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 phosphorylated when placed at a tyrosine kinase recognition site is a satisfying  - 156 demonstration of the specificity of the pl30  gag-fps protein kinase  activity for tyrosine residues. While i t seems probable that the amino acids surrounding tyrosine-1073 of wt P  13 gag-fps 0  and other tyrosines 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 i s a strict requirement for tyrosine at the site of phosphorylation. It i s 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 i s an obvious sequence relationship between the enzymatic domains of  tyrosine protein kinases such as FSV pl30  gag-fps and those  kinases specific for serine and threonine residues, such as the catalytic 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 i s 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 pl30  gag-fps  may be folded such that only a Substitutions tyrosine signal formed at residue by at the position 1073 surrounding can1073 be positioned recognition might alsotosequence. disrupt accept a aphosphate conformational group.  - 157 -  Amino acid substitutions residue of P 1 3 0  gag  ~  fps  at the conserved tyrosine-1073  did not completely abolish i t s activity.  I  have changed tyrosine-1073 to serine, threonine, glycine and phenylalanine 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. PlSO^g -^ -  In addition, these transformed cells expressed  with a similar reduced biochemical activity.  8  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 considering 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 l e x i b i l i t y into the polypeptide. These considerations  imply  that  the  important  characteristic  tyrosine-1073 is i t s ability to become phosphorylated. fore concluded that the reduced kinase activity  of  I have there-  and transforming  potential of the mutant proteins are not due to disruptive changes in protein conformation, but are a consequence of the absence of phosphorylation.  Although tyrosine-1073 appears to be important for regulation  of  pisogag-^P  8  tyrosine  kinase  activity,  there  the are  - 158 -  additional tyrosine, serine and threonine phosphorylation sites within the PISO - "^ 83  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,  specifities.  each  with  different  amino  acid  substrate  - 159 -  CHAPTER 6  6.0 Site-directed Mutagenesis of Lysine-950 Within PlSQgag"^  8  Eliminates Both Its Kinase Activity and Transforming Ability.  6.1  Introduction  To understand more fully the biological roles of tyrosine protein kinases, i t i s important to identify the specific structural domains or amino acid residues that contribute to the enzymatic activities of these proteins. P140 ~"^ gag  ps  The catalytic domains of  p60 , src  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). cleavage fragments possess tyrosine kinase activity,  Since these 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  src  tyrosine  protein kinase are distantly related, based on sequence homology within their respective catalytic domains.  More specifically, this homology  includes a lysine residue that i s proposed to function in ATP-binding.  - 160 -  The identity  and putative  function of  this  lysine was  determined by studies using the reactive ATP analogue, sulfonylbenzoyl 5' - adenosine (FSBA).  p-fluoro-  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 i s  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 6.1).  (Figure  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 . ,  et a l . , 1984).  1981)  and  lysine-295  within  p60  src  (Kamps,  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 t s 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-fluorosulfonylbenzoyl5'-Adenosine (FSBA) and Adenosine 5'-triphosphate (ATP).  Adenine  CHo  FSBA  .0  OH OH  CH  Adenine  9  .0.  OH OH  ATP  - 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  src  tyrosine protein kinase a l l orient  a homologous lysine residue such that i t reacts with FSBA. These finding provide strong evidence that the sequence homology found between p60  src  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  P130  g a g - f p s  contains a lysine at position 950 which i s homologous to the highly conserved lysine-295 of p 6 0  src  (Shibuya and Hanafusa, 1982), which  has been proposed to function as an ATP binding site in (Kamps, et a l . , 1984).  p60  src  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 ~ " . gag  fps  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.  Zoller, lysine-950 within P 1 3 0  In  collaboration  with  M.  has been mutated in order to  g a g _ f p s  evaluate the contribution of this residue in the phosphotransfer reaction.  If  the kinase activity  intrinsic  to P 1 3 0  g a g -  ^  is  p s  crucial for i t s transforming function, then alterations in i t s 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 " P Kinase Activity by Treatment gag  f  s  with FSBA  In  order  to  measure  the  inactivation  of  Pl40  gag  ~^  ps  kinase activity by FSBA over time, immune-precipitates prepared from FSV-L5 transformed CEF were incubated at 37°C with 1.7 mM FSBA.  Ali-  quotes were removed at timed intervals following the addition of FSBA, placed  on  ice,  incubated  in  the  described in section 2.4 and the  presence  of  f£- P]ATP 32  as  P-labelled proteins resolved by  SDS-PAGE (Figure 6.2A, lanes 1-6).  To control for nonspecific loss of  P140  during  g a g - f p s  P140 ~^ gag  ps  kinase  activity  immune-precipitates were  not  incubation treated  with  at FSBA,  37 °C, but  were incubated at 37°C, sampled at the start and finish of the experiment and assayed as for the FSBA treated samples (Figure 6.2A; lanes 7  - 165 Figure 6.2  Inactivation treatment  of  with  pi40 FBSA  g a g  ~  kinase  f p s  A.  P140gag-fps kinase activity  FSBA  activity  by  inactivation  of  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  activity for FSBA inactivation:  gag  ~^  ps  kinase  Immunoprecipitates from  FSV-L5 transformed cells were incubated in the presence of various concentrations of FSBA at 37 °C for 60 minutes, kinased, and analysed by 7.5% SDS-PAGE followed by autoradiography.  Lanes 1, immunoprecipitates untreated  with FSBA and incubated on ice for 60 minutes. through 8 represent  Lanes 2  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 t s 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). from  To quantitate the extent of enzyme inactivation resulting  treatment  of  Pi40  gag  ~P f  s  with  FSBA,  32  P-labelled  teins were excised from the gel and the amount of was determined by Cerenkov counting.  32  pro-  P-incorporated  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 S~ P ga  that had not been treated with FSBA. decrease  in  P  i40  g a g - f  P  s  kinase  attributed to FSBA inactivation.  f  s  This represents a 4.3-fold  activity  that  can probably  be  The inactivation of CAT by FSBA was  not quantitated since there was an obvious and significant decrease in oo  incorporation of FSBA.  Therefore,  into histones following treatment of CAT with pre-incubation of p i 4 0  g a g  "  with 1.7 mM FSBA  f p s  decreased the phosphotransferase activity in a time-dependent fashion. To demonstrate that the loss in P i 4 0  gag-  ^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  gag-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 concentrations (Figure 6.2B; lanes 2 through 8). untreated P l 4 0  g a g  ~  containing  f p s  Lane 1 represents an  immune-precipitate that  on ice during the incubation period.  was  left  The results indicate that an  increase in FSBA concentration can be correlated with a decrease in kinase activity,  which suggests that FSBA inactivates  P140  g a g - : f p s  by reacting with a portion of the protein that functions in ATP binding.  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  gag  ~  fps  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 inactivation 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 P140 ~-^P gag  binding site that may be modified by FSBA.  S  contains an ATP  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 tion in ATP binding.  g a g - f p s  Lysine-950 within PISO  ,  69-6  proposed to func"^  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 t s side chain is also positively charged.  However, i f the size and shape of lysine-950 is  absolutely c r i t i c a 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 t s 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 .  Oligonucleo-  s  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'  LYS Wild-type FSV  5 * HXGTGGCGGTGAGATCC-3'  ARG  FSV-R (950)  5 -CCGTGGCGGTGGGATCC-3 *  GLY  FSV-G (950)  5' -CXXTGGCGGTGAMTCC-3 *  LYS  FSV-K (950)  1  - 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 glycine.  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 nucleotides 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 oligonucleotide 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 transforming 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 containing transformed cells with a round refractile morphology were obvious  - 173 -  on a background of normal flat rat-2 cells.  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  transfection with wtFSV or revertant  cells  that  appeared  after  FSV-K(950) DNAs were picked,  cloned in soft agar, and expanded in mass culture.  The morphological  phenotype of the revertant transformed cell lines was indistinguishable from that of the wild-type FSV cell line (Figure 6.4; panel B and D), and both of these FSV transformed c e l l lines formed large colonies in soft agar (data not shown).  As expected, the cells cloned from these  two FSV transformed cell lines were very round and refractile, unlike the normal rat-2 cells which were characteristically very flat 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 p gag-fps 130  I with an alternative  cotransfected the mutated FSV genomes  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 transfection 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  cells;  and panel D,  transformed cells.  revertant  FSV-K  (950)  - 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 c e l l s , 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  selected and tested for expression of P 1 3 0 of cells were labelled with [ ^S]methionine 3  FSV-G(950) g a g - f p s  .  Equal numbers  for 16 hours,  immunoprecipitated with  anti-pl9  presence of P 1 3 0  by SDS-PAGE and fluorography.  g a g - f p s  gag  DNAs were  serum and examined  lysed,  for  the  Of the 15  different clones tested from each set of FSV-R(950) or FSV-G(950) transfected cells, only one clone from each set was found to express piSOgag-fP  8  (Figure 6.5; panel A).  The figure also shows two TK  positive rat-2 lines [R-2(tk)] which are negative for expression and a wt FSV P 1 3 0 The cells  expressing  g a g  FSV-R(950)  "  f p s  P130  g a g _ f p s  expressing c e l l line (K-950).  and FSV-G(950)  PISO^"^  8  pro-  - 177 Figure 6.5:  Analysis of wild-type and mutant FSV transfected rat-2 cells  for  FSV  88,8  PiaO ""*  protein kinase activity.  138  synthesis  and  tyrosine  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 cells. R-2 indicates normal rat-2 cells.  A. Rat-2 cells cotransfected with pTK-^  plus mutant FSV-R(950) or FSV-G(950) DNAs were selected in HAT-DMEM. wells,  HAT resistant clones were grown in 35 mm  labelled for  methionine,  16 hours with 100 uCi  immunoprecipitated  with  [ S]35  anti-pl9  gag  serum and examined by gel electrophoresis and fluorography 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 MnCl  2  30°C.  and 2.5 uCi  [ $- P]ATP 32  for  15 minutes  at  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 flat 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 pl30  gag-fps proteins  had kinase activity, wild type FSV and mutant [FSV-R(950) or FSV-G (950)]  P130  proteins  g a g - f p s  were  immunoprecipitated  from equal  numbers of unlabelled cells and the immune complexes were incubated with  MnCl , 2  [)(- P]ATP, 32  and  enolase (Figure 6.5; panel C).  soluble  denatured  rabbit  muscle  In this reaction wtFSV pl30  gag-fps  (K-950) i s autophosphorylated and also phosphorylates enolase • at a single tyrosine residue (Cooper, et a l . , 1984a). the mutant c e l l autophosphorylation exogenous  lines of  However, neither of  (R-950 nor G—950) demonstrated detectable P130  substrate enolase;  or  g a g - f p s  phosphorylation  of  the background from both of  the these  reactions i s similar to that found with normal rat-2 cells (Figure 6.5; panel C). lysine-950  This experiment indicates that alterations in the codon for within pl30  gag-fps yields  tyrosine protein kinase. In FSV-transformed  an  enzymatically  inactive  cells, P13 gag-fps 0  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)  P130  was phosphorylated in cells  g a g - f p s  expressing this protein, both normal rat-2 cells (R-2) and mutant FSV-R (950) rat-2 cells  were metabolically labelled with  (R-950)  orthophosphate for 12 hours,  3 2  lysed, immunoprecipitated with a n t i -  piggag serum, and the labelled proteins resolved by SDS-PAGE. data show that mutant R-950  P-  P130  g a g  ~  : f p s  The  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 protein,  purified  in vivo  32  P-labelled  FSV-R(950)  was subjected to tryptic peptide analysis. (950)  P130  g a g  ~^  tryptic  p s  g a g  ~  f p s  Inspection of the FSV-R-  map indicates that  it  is missing the  major site of tyrosine phosphorylation, tyrosine-1073 panel C).  P130  (Figure 6.6;  This site is contained within tryptic phosphopeptides 3b and  3c of wtFSV (950) P 1 3 0  PlSCr g a g - f p s  peptide map of  5 8 6  ^^  (Figure  6.6,  panel  In fact, the FSV-R  A).  tryptic map i s similar to the tryptic phosphothe mutant FSV-S(1073)  P130  g a g _ f p s  (Figure  6.6;  panel B) in which the major tyrosine phosphoaceptor site (tyrosine1073) has been destroyed (Chapter 5 ) . P130  g a g  ~  f p s  Interestingly,  FSV-R (950)  appears to be phosphorylated at tryptic phosphopeptide  4, which represents the other major tyrosine site which is phosphorylated exclusively in vivo in both wt FSV  P130  g a g - f p s  and in a l l  - 181 -  Figure 6.6:  Comparison gag-fps  of  tryptic  phosphopeptides  from  pl30  encoded  by  wild-type  FSV-S(1073) and mutant FSV-R (950).  FSV,  mutant  Cells were labelled  op  with 1 mCi  P-orthophosphate for  immunoprecipitated  12 hours,  lysed,  and pl30  gag-fps was  identified  as described in the legend for Figure 6.5. Gel-purified PISO^^-^P  8  was  then  digested  with  trypsin  and  separated by electrophoresis at pH2.1 on thin-layer cellulose plates in the f i r s t dimension and chromatography 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 P I S O ^ ^ 89  -  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 l e 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 phosphorylation.  However, spot 5,  and probably spot 8, contain phospho—  serine, and spot 6 i s known to be a minor site of tyrosine phosphorylation (Chapter 3).  Presumably the majority of these uncharacterized sites of phosphorylation contain phosphoserine, and possibly threonine, P130  since  g a g - f p s  phosphoamino  acid  analysis  some phosphoof  FSV-R(950)  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 phosphothreonine, confirming that this mutant protein i s 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  (950)  P130  32  g a g _ f p s  P-labelled have  FSV-S(1073)  P130  similar mobilities  indicates that FSV-R(950) P I S O ^ " ^ 6  8  g a g _ f p s  (Figure  and 6.6).  FSV-R This  is only missing the sites of  autophosphorylation consistent with i t s lack of kinase activity when  - 184 assayed in the immiine complex reaction (Figure 6.5, panel C). that  FSV-  R(950) P l 3 0  related to gag-fps  both  contained sites  g a g - f p s  wtFSV  pi30  and  g a g - f p s  of  The fact  phosphorylation  mutant  FSV-S(1073)  pl30  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) protein  suggests  affect  that  changes at  the protein's  ability  position  950 within  P130  g a g - f p s  to function in the phosphotransfer  reaction. 6.2.5  Trans-Phosphorylation of FSV-R(950) P 1 3 0 FSV P 1 4 0  g a g  -  gag  ~  fps  by  f p s  Since tyrosine-1073 i s the major site of autophosphorylation i t is not surprising that this site is not phosphorylated in an enzymatically  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)  P130  could  g a g - f p s  be phosphorylated  enzymatically active FSV P 1 4 0  g a g - f p s  .  in trans  by  the  Cleared c e l l lysates from  FSV-L5 transformed rat-1 cells and FSV-R(950) rat-2 cells were prepared. One-half of each lysate was combined and the mixture was immunoprecipitated, while the remainder of each lysate was immunoprecipitated separately.  The precipitated immune complexes were kinased in  the presence of MnCl  2  and [ - P]ATP 32  at 20°C for  15 minutes,  - 185 -  following which the  P-labelled proteins were separated by SDS-  PAGE and visualized by autoradiography. in vitro  For comparative controls,  kinase reactions of wtFSV P130  gag-fps and mutant FSV-  S(1073) P 1 3 0  g a g - f  P  were also separated on the same gel, while a  s  kinased rat-2 immune-precipitate was included as a background control (Figure 6.7). When were  FSV  pi40  coimmunoprecipitated  [)(- P]ATP  both proteins  32  lane 1).  g a g - f  P  and  s  and  incubated  were  labelled with  However FSV-R(950) P I S O ^ ^ P  same ATP:phosphotransferase kinased alone  FSV-R(950)  reaction  in  pi30  the 3 2  P  g a g _ f  presence  P  s  of  (Figure 6.7;  was not labelled in the  8  when  immunoprecipitated and  (Figure 6.7; lane 3), while FSV p i 4 0  labelled under the same conditions (Figure 6.7; lane 2).  g a g  ~  f p s  was  These data  qp  indicate  that  P130 ~^P gag  s  the  incorporation  pl30  P140  gag-fps  P  into  order  to  that  were  t h e  32  g a g  "P . f  s  identify  the  sites  phosphorylated  p-iabelled  protein  within in trans  was  phosphopeptides  found  in  FSV-R(950) by  excised,  concentrated and subjected to tryptic peptide analysis. tryptic  FSV-R(950)  i s a result of trans-phorphorylation by the enzymatic  activity intrinsic to FSV p i 4 0 In gag-fps  of  FSV  eluted, The major  FSV-R(950) P130  gag-fps phos-  phorylated in trans were those which correspond to the major autophosphorylation site of wtFSV p i 3 0  gag  ~P f  s  (Figure 6.8; 3a and 3b),  which i s known to contain tyrosine-1073 (Chapter 3 and 4).  These data  - 186 -  Figure 6.7:  In vitro  phosphorylation  by FSV P l 4 0  g a g _ f  P . s  of  Cell  FSV-R(950)  pi30  g a g - f  P  s  lysates from unlabelled  cells were either immunoprecipitated together or separately as indicated below, kinased in the presence of 10 mM MnCl  2  plus  5 uCi  [^- P]ATP 32  and analysed  by  electrophoresis through a 7.5% polyacrylamide gel f o l lowed by autoradiography.  The kinase reactions were  from the following immunoprecipitated cell Lane 1, FSV-L5 ( P 1 4 0 ~ ) gag  FSV-R(950) (Pl40 rat-2  gag_f  rat-2 P ) s  cells;  fps  transformed cells plus  cells;  transformed cells; lane 4, wt  lysates:  FSV  lane  2,  FSV-L5  lane 3, FSV-R(950) (PISO^^P ) 53  trans-  formed cells; lane 5, FSV-S(1073) transformed cells; lane 6, normal rat-2 cells.  -  187  -  1 2 34 5 6 P140 P130 P130  - 188 -  Figure 6.8:  Tryptic phosphopeptide analysis of in vitro phosphorylated  wt  FSV  f  pi30^^~ P  phosphorylated P140 g~ P ga  (950)  f  P130  in vitro  FSV-R(950)  encoded  s  and  s  gag_f  P  s  by  P130 ~ P . gag  FSV-L5  were  trans-  and  mutant  coimmunoprecipitated  f  s  FSV-R and  labelled in an immune complex kinase assay following immunoprecipitation  with  anti-p 19  gag serum  from  FSV-L5 transformed cells and FSV-R(950) rat-2 cells, wt FSV P 1 3 0 ~ from wt FSV transformed cells was immunoprecipitated and kinased in an identical fashion. 32 gag  The  fps  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: (950) pi30 Pl^gag-fps.  g a g _ f  P  A, wt FSV P 1 3 0 ~ P ; gag  s  f  trans-phosphorylated  s  B,  FSV-R  by  FSV  - 190 suggest  that  the mutant FSV-R(950)  P130  g a g _ f p s  has maintained a  conformation around the major tyrosine phosphoacceptor site which permits 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-  fically inactive the kinase activities associated with the cyclicnucleotide dependent protein kinases cAPK (Zoller, et a l . , 1981) and cGPK (Hashimoto, et a l . , 1982), and the tyrosine specific protein kinase  p60  (Kamps,  src  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. the FSV P130 ~"^ gag  ps  Inspection of  amino acid sequence indicates that lysine-950  i s the likely 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 . src  Although  the FSBA inactivation  data demon-  strates the presence of an ATP binding site within P 1 3 0  gag  ~ , a fps  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 i s 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). sequence surrounding  A comparison of the amino acid  lysine-950 of pl30  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 i s 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 i s 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 dehydrogenases, 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 l e x i b i l i t y 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) i s 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 homologous position (Wierenga and Hoi, 1983).  It i s interesting to note  that this array of glycines i s also found in the GTP binding protein of p21 ~ , c  ras  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 pl30  gag-fps belongs to a  family of tyrosine kinases that are a l l related to p 6 0  src  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 i s 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 i s 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 an essential role in catalysis. site-directed mutagenesis of  g a g - f p s  plays  As more direct evidence, the  the P 1 3 0  g a g _ f p s  lysine-950 destroys  the tyrosine protein kinase activity intrinsic to FSV P 1 3 0 ~ ^ . gag  ps  In addition, mutations of lysine-950 also eliminate the transforming activity of PISO ^ ^^ and support the idea that tyrosine protein 69  -  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  The P130  g a g - f p s  g a g - f p s  .  stable  expression  of  the  mutant  FSV-R(950)  protein, with an arginine substituted for lysine-950  and the ability 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)  is  PISO^^'1^  similar  to  other  FSV  P130  g a g - f p s  proteins which have mutations at residue 1073. Since tyrosine-1073 is normally  the  major  site  P1306ag-fps both in vitro lack P130  of  phosphorylation  g a g - f p s  phorylation  of  tyrosine  phosphorylation  (autophosphorylation) at  tyrosine-1073  within  and in vivo, within  the  FSV-R(950)  i s consistent with i t s inability to undergo autophosor  to phosphorylate  the  exogenous  substrate enolase  in vitro.  P130  g a g - f p s  isolated from FSV transformed cells is phos-  phorylated not only at tyrosine, but also at serine and threonine r e s 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)  13Qgag-fps indicated that this mutant protein was phosphorylated  P  at tyrosine residues in intact cells.  The phosphorylation of tyrosine  residues within FSV-R(950) P 1 3 0  ,  g a g _ f p s  which i s devoid of  tyro-  sine protein kinase activity, suggests that this protein may be phosphorylated by a cellular protein kinase specific for tyrosine residues. On the  other  hand,  the  possibility  exists  that  phosphorylation detected in the FSV-R(950) P^Cp^'^ product of a unique type of autophosphorylation.  the  tyrosine-  may be the  - 196 -  CHAPTER 7 7.0 SUMMARY  The transforming protein of FSV has an intrinsic tyrosine specific protein kinase activity and i s itself phosphorylated at multiple tyrosine and serine residues.  Thus, phosphorylation of the FSV  transforming protein i s complex and may well affect i t s activity and function.  Since the kinase and transforming activities of FSV  P140  are related,  g a g _ f p s  I  have investigated the phosphorylation  of the FSV transforming protein in detail.  The P140  g a g _ f p s  ,  major which  fps-specific include  two  phosphorylation  sites  phosphotyrosine  residues  of and  FSV 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 i s 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 - "^ , 53  6  8  tyrosine-1073.  Oligonucleotide-directed mutagenesis was used to change the codon for  - 197 -  tyrosine-1073 to a codon for phenylalanine, glycine. P130  serine,  threonine or  These amino acid substitutions at position 1073 within  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. mutant  transformed  cells  In addition these different  expressed pl30  gag-fps proteins  reduced kinase activities.  with  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 i s i t s ability to become phosphorylated.  Therefore, given the caveat that  mutations at residue 1073 do not have a global effect on protein conformation, I have interpreted the reduced kinase activity and transforming 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 phosphorylated when placed at a tyrosine kinase recognition site demonstrates the specificity of the p gag-fps protein kinase activity for 130  - 198 -  tyrosine residues. surrounding  While i t seems probable that the amino acids  tyrosine-1073 of  wtFSV P 1 3 0  gag  ~  fps  are  important  in  targeting the kinase to a particular residue, i t i s apparent that there i s a strict requirement for tyrosine at the site of phosphorylation. Amino acid substitutions at tyrosine-1073 of did not completely abolish i t s activity.  Pl30  g a g _ f  P  s  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  gag-f  P  s  other than  tyrosine phosphorylation i s involved in i t s transforming ability. Although, these data strongly suggest that tyrosine phosphorylation of PISO^^-^P  8  can modulate i t s activity and strengthen the case for  the involvement of tyrosine phosphorylation in transformation by FSV.  Mutations P130  the  putative  ATP-binding  site  of  fps  at lysine-950 destroy both i t s kinase and transforming  activities.  These results support the idea that the tyrosine kinase  gag  ~  within  activity intrinsic to p i 3 0 function.  g a g - f  P  s  i s essential for i t s tranforming  A conservative amino acid change to arginine at residue 950  allowed for the stable expression of the mutant protein. Tryptic phosphopeptide 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 ~ P gag  f  s  as a site  exclusively  phosphorlyated  in vivo.  The  - 199 -  phosphorylation of tyrosine residues within a mutant protein devoid of intrinsic pl30  tyrosine  protein  kinase  activity  suggested  that  FSV  gag-fps may be a target  for  phosphorylation  protein kinase specific for tyrosine residues.  by a  cellular  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 i s consistent with many investigations to date which reveal that cellular transformation by FSV occurs by a complex  and as yet  undefined mechanism.  Nonetheless,  the use of  oligonucleotide-directed mutagenesis to specifically alter key amino acid residues within FSV P 1 3 0  g a g - f p s  provides a powerful technique  that may help to unravel this complex mechanism.  - 200 REFERENCES Abrams, H.D., Rohrschneider, L.R., Eisenman, R.N. (1982). 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