A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.     

A major site of tyrosine phosphorylation within the SH2 domain of Fujinami sarcoma virus P130gag-fps is not required for protein-tyrosine kinase activity or transforming potential.

Phosphorylation of the major autophosphorylation site (Tyr-1073) within Fujinami sarcoma virus P130gag-fps activates both the intrinsic protein-tyrosine kinase activity and transforming potential of the protein. In this report, a second site of autophosphorylation Tyr-836 was identified. This tyrosine residue is found within a noncatalytic domain (SH2) of P130gag-fps that is required for full protein-kinase activity in both rat and chicken cells. Autophosphorylation of this tyrosine residue implies that the SH2 region lies near the active site in the catalytic domain in the native protein and thus possibly regulates its enzymatic activity. Four mutations have occurred within the SH2 domain between the c-fps and v-fps proteins. Tyr-836 is one of these changes, being a Cys in c-fps. Site-directed mutagenesis was used to investigate the function of this autophosphorylation site. Substitution of Tyr-836 with a Phe had no apparent effect on the transforming ability or protein-tyrosine kinase activity of P130gag-fps in rat-2 cells. Mutagenesis of both autophosphorylation sites (Tyr-1073 and Tyr-836) did not reveal any cooperation between these two phosphorylation sites. The implications of the changes within the SH2 region for v-fps function and activation of the c-fps oncogenic potential are discussed.     

The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.     

Localization and characterization of phosphorylation sites of the Fujinami avian sarcoma virus and PRCII virus transforming proteins

Fujinami sarcoma virus (FSV) and PRCII are avian sarcoma viruses which share cellularly derived v-fps transforming sequences. The FSV P140gag-fps gene product is phosphorylated on three distinct tyrosine residues in transformed cells or in an in vitro kinase reaction. Three variants of FSV, and the related virus PRCII which lacks about half of the v-fps sequence found in FSV, encode gene products which are all phosphorylated at tyrosine residues contained within identical tryptic peptides. This indicates a stringent conservation of amino acid sequence at the tyrosine phosphorylation sites which presumably reflects the importance of these sites for the biologic activity of the transforming proteins. Under suitable conditions the proteolytic enzymes p15 and V8 protease each introduce one cut into FSV P140, p15 in the N-terminal gag-encoded region and V8 protease in the middle of the fps-encoded region. Using these enzymes we have mapped the major site of tyrosine phosphorylation to the C-terminal end of the fps region of FSV P140gag-fps. A second tyrosine phosphorylation site is found in the fps region of FSV P140 isolated from transformed cells, and a minor tyrosine phosphorylation site is found in the N-terminal gag-encoded region. Our results suggest that the C-terminal fps-encoded region is required for expression of the tyrosine-specific protein kinase activity.     

Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues

A number of oncogenic viruses encode transforming proteins with protein kinase activities apparently specific for tyrosine residues. Recent evidence has raised questions as to the substrate specificity of these kinases in general and the physiological relevance of tyrosine phosphorylation in particular. The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) is strongly phosphorylated at 2 tyrosine residues in FSV-transformed cells of which 1 (Tyr-1073) is also the major site of P130gag-fps intermolecular autophosphorylation in vitro. We have investigated the specificity of the protein kinase activity intrinsic to FSV P130gag-fps by using site-directed mutagenesis to change the codon for Tyr-1073 to those for the other commonly phosphorylated hydroxyamino acids, serine and threonine. This approach has some advantages over the use of synthetic peptides to define protein kinase recognition sites in that the protein containing the altered target site can be expressed in intact cells. In addition it allows higher order as well as primary structure of the enzyme recognition site to be considered. Neither serine nor threonine were phosphorylated when substituted for tyrosine at position 1073 of P130gag-fps indicating a stringent specificity for tyrosine as a substrate of the P130gag-fps protein kinase autophosphorylating activity. Consistent with the suggestion that tyrosine phosphorylation is of functional significance we find that these and other FSV Tyr-1073 mutants have depressed enzymatic and oncogenic capacities.     

Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5''-fps sequence has a role in the transforming activity.

Fujinami sarcoma virus (FSV) genome codes for the gag-fps fusion protein FSV-P130. The amino acid sequence of the 3' one-third portion in v-fps is partially homologous to the 3' half of pp60src, or the kinase domain, but the sequence of the 5' portion is unique to v-fps. To identify a possible domain structure in the v-fps sequence responsible for cell transformation, we constructed various deletion mutants of FSV with molecularly cloned viral DNA. Their transforming activities were assayed by measuring focus formation on chicken embryo fibroblasts and rat 3Y1 cells and tumor formation in chickens. The mutants carrying a deletion at the 3' portion in v-fps, the kinase domain, lost transforming activity. The mutants carrying an approximately 1-kilobase deletion within the 5' portion of the v-fps sequence retained focus-forming activity and tumorigenicity in the chicken system, but the efficiency of focus formation was about 10 times lower than that of the wild type. The morphology of these transformed cells was distinct from that observed in cells infected with wild-type FSV. Furthermore, these mutants could not transform rat 3Y1 cells, although wild-type FSV DNA transformed rat 3Y1 cells at a high frequency. The mutants carrying a larger deletion in the 5' portion of fps completely lacked the transforming activity. These results suggest that the 3' portion of the v-fps sequence is necessary but not sufficient for cell transformation and that the 5' portion of v-fps has a role in the transforming activity.     

A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity.

The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation.     

The myristylation signal of p60v-src functionally complements the N-terminal fps-specific region of P130gag-fps.

The P130gag-fps protein-tyrosine kinase of Fujinami sarcoma virus contains an N-terminal fps-specific domain (Nfps) that is important for oncogenicity. The N-terminal 14 amino acids of p60v-src, which direct myristylation and membrane association, can replace the gag-Nfps sequences of P130gag-fps (residues 1 to 635), producing a highly transforming src-fps polypeptide. Conversely, gag-Nfps can restore modest transforming activity to a nonmyristylated v-src polypeptide. These results emphasize the modular construction of protein-tyrosine kinases and indicate that Nfps, possibly in conjunction with gag, functions in the subcellular localization of P130gag-fps.     

Mapping of multiple phosphorylation sites within the structural and catalytic domains of the Fujinami avian sarcoma virus transforming protein.

The phosphorylation sites of the P140gag-fps gene product of Fujinami avian sarcoma virus have been identified and localized to different regions of this transforming protein. FSV P140gag-fps isolated from transformed cells is phosphorylated on at least three distinct tyrosine residues and one serine residue, in addition to minor phosphorylation sites shared with Pr76gag. Partial proteolysis with virion protease p15 or with Staphylococcus aureus V8 protease has been used to generate defined peptide fragments of P140gag-fps and thus to map its phosphorylation sites. The amino-terminal gag-encoded region of P140gag-fps contains a phosphotyrosine residue in addition to normal gag phosphorylation sites. The two major phosphotyrosine residues and the major phosphorserine residue are located in the carboxy-terminal portion of the fps-encoded region of P140gag-fps. P140gag-fps radiolabeled in vitro in an immune complex kinase reaction is phosphorylated at only one of the two C-terminal tyrosine residues phosphorylated in vivo and weakly phosphorylated at the gag-encoded tyrosine and at a tyrosine site not detectably phosphorylated in vivo. Thus, the in vitro tyrosine phosphorylation of P140gag-fps is distinct from that seen in the transformed cell. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three Fujinami avian sarcoma virus variants showed that the phosphotyrosine-containing peptides are invariant, and this high degree of sequence conservation suggests that these sites are functionally important or lie within important regions. The P105gag-fps transforming protein of PRCII avian sarcoma virus lacks one of the C-terminal phosphotyrosine sites found in Fujinami avian sarcoma virus P140gag-fps. Partial trypsin cleavage of FSV P140gag-fps immunoprecipitated with anti-gag serum releases C-terminal fragments of 45K and 29K from the immune complex that retain an associated tyrosine-specific protein kinase activity. This observation, and the localization of the major P140gag-fps phosphorylation sites to the C-terminal fps region, indicate that the kinase domain of P140gag-fps is located at its C terminus. The phosphorylation of P140gag-fps itself is complex, suggesting that it may itself interact with several protein kinases in the transformed cell.     

The human c-fps/fes gene product expressed ectopically in rat fibroblasts is nontransforming and has restrained protein-tyrosine kinase activity.

A 13-kilobase EcoRI genomic restriction fragment containing the human c-fps/fes proto-oncogene locus was expressed transiently in Cos-1 monkey cells and stably in Rat-2 fibroblasts. In both cases, human c-fps/fes directed synthesis of a 92-kilodalton protein-tyrosine kinase (p92c-fes) indistinguishable from a tyrosine kinase previously identified with anti-fps antiserum which is specifically expressed in human myeloid cells. Transfected Rat-2 cells containing approximately 50-fold more human p92c-fes than is found in human leukemic cells remained morphologically normal and failed to grow in soft agar. Synthesis of p92c-fes in this phenotypically normal line exceeded that of the P130gag-fps oncoprotein in a v-fps-transformed Rat-2 line. Despite this elevated expression, human p92c-fes induced no substantial increase in cellular phosphotyrosine and was not itself phosphorylated on tyrosine. In contrast, p92c-fes immunoprecipitated from these Rat-2 cells or expressed as an enzymatically active fragment in Escherichia coli from a c-fps/fes cDNA catalyzed tyrosine phosphorylation with an activity similar to that of v-fps/fes polypeptides. Thus, p92c-fes is not transforming when ectopically overexpressed in Rat-2 fibroblasts. This lack of transforming activity correlates with a restriction imposed on the kinase activity of the normal c-fps/fes product in vivo which is apparently lifted for v-fps/fes oncoproteins, suggesting that regulatory interactions within the host cell modify fps/fes protein function and normally restrain its oncogenic potential.     

Enzymatic activation of Fujinami sarcoma virus gag-fps transforming proteins by autophosphorylation at tyrosine.

Site-directed mutagenesis of the Fujinami sarcoma virus (FSV) genome has suggested that Tyr 1073 of the P130gag--fps protein-tyrosine kinase is a regulatory site. To investigate directly the ability of tyrosine phosphorylation to affect P130gag--fps kinase activity, the phosphotyrosyl phosphatase inhibitor orthovanadate and partially purified phosphotyrosyl phosphatases were used to manipulate the stoichiometry of P130gag--fps phosphorylation. Phosphorylation of P130gag--fps at Tyr 1073 correlated with enhanced kinase activity. The thermolabile phosphorylation, kinase activity and transforming ability of P140gag--fps encoded by a temperature-sensitive (ts)FSV variant were restored at the non-permissive temperature for transformation by incubation of infected cells with orthovanadate. In this case tyrosine phosphorylation can apparently functionally reactivate a conditionally defective v-fps kinase activity. These data suggest that reversible autophosphorylation at a conserved tyrosine within the v-fps kinase domain is a positive regulator of enzymatic activity and biological function. Phenotypic suppression of the tsFSV genetic defect by orthovanadate emphasizes the potential importance of phosphotyrosyl phosphatases in antagonizing tyrosine kinase action. It is suggested that autophosphorylation may constitute a molecular switch by which some protein-tyrosine kinases are activated.     

Investigation of the role of P130gag-fps in transformation: generation and use of a temperature-sensitive mutant P130gag-fps.

Changing Glu-1025 to Asp in Fujinami sarcoma virus P130gag-fps made the protein temperature sensitive for transformation and protein-tyrosine kinase activity. Another mutant, Phe-1073 P130gag-fps, lacking the major autophosphorylation site, has an extended latent period for transformation (G. A. Weinmaster, M. J. Zoller, M. Smith, E. Hinze, and T. Pawson, Cell 37:559-568, 1984). By introducing the Asp-1025 lesion into Phe-1073 P130gag-fps, we showed that this mutant protein is required for the maintenance of the transformed phenotype of Phe-1073 P130gag-fps-expressing cells.     

Nucleotide sequence of v-fps in the PRCII strain of avian sarcoma virus.

PRCII is an avian retrovirus whose oncogene (v-fps) induces fibrosarcomas in birds. The viral gene v-fps arose by transduction of an undetermined portion of a cellular gene known as c-fps. PRCII is weakly oncogenic when compared with Fujinami sarcoma virus, another transforming virus containing v-fps. As a first step in the elucidation of the molecular basis for the decreased virulence of PRCII, we have determined the entire nucleotide sequence of v-fps in the PRCII genome. The v-fps domain in PRCII encodes a polypeptide with a molecular weight of ca. 60,500 fused to a portion of the polyprotein encoded by the viral structural gene gag. The hybrid gag-fps polyprotein of PRCII would have a molecular weight of ca. 98,100, in accord with results of previous studies of the protein encoded by the PRCII genome. The leftward junctions between fps and gag in Fujinami sarcoma virus and PRCII are located at the same position in fps, but at different positions in gag. A sequence of 1,020 nucleotides, bounded by direct repeats of 6 nucleotides, is present in v-fps of Fujinami sarcoma virus but absent from PRCII. Our data should permit further explorations of the relationship between structure and function in the transforming protein encoded by v-fps.     

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.     

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.     

Activation of the transformation potential of the cellular fps gene

Chicken cellular-fps (c-fps) sequences were substituted for viral-fps (v-fps) sequences in two retroviral genome structures, one that expressed a c-fps gene product that was indistinguishable from the normal c-fps gene product expressed in chicken bone marrow cells, and another that expressed a gag-fps fusion protein. When c-fps gene sequences (without linked gag gene sequences) were expressed at high levels in a viral vector, no transformation of fibroblasts was detected. It was previously demonstrated that the corresponding v-fps sequences could transform fibroblasts. When the same c-fps sequences were expressed in a form linked to gag gene sequences, transformation of fibroblasts and induction of tumors were observed. The data suggest that the c-fps gene product lacks transformation potential by itself even when overexpressed and that the transformation potential of the c-fps gene can be activated by either mutation (or mutations) in the fps coding region or by fusion with viral gag gene sequences.     

Mutagenesis of Fujinami sarcoma virus: evidence that tyrosine phosphorylation of P130gag-fps modulates its biological activity

The 130 kd transforming protein of Fujinami sarcoma virus (FSV P130gag -fps) possesses a tyrosine-specific protein kinase activity and is itself phosphorylated at several tyrosine and serine residues in FSV-transformed cells. We have used oligonucleotide-directed mutagenesis of the FSV genome to change the TAT codon for tyrosine (1073), the major site of P130gag -fps phosphorylation, to a TTT codon for phenylalanine that cannot be phosphorylated. This mutant FSV induces the transformation of rat-2 cells but with a long latent period as compared with wild-type FSV. The P130gag -fps protein encoded by the mutant retains the ability to phosphorylate tyrosine, but is five times less active as a kinase in vitro than wild-type FSV P130gag -fps. These data indicate that tyrosine phosphorylation stimulates the biochemical and biological activities of FSV P130gag -fps, and they set a precedent for the ability of this amino acid modification to modulate protein function.     

Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.     

Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases

The receptors for polypeptide growth factors and proteins coded by oncogenes of the src family are endowed with protein kinase activity and share the uncommon property of autophosphorylating at tyrosine residues. It is unclear whether the tyrosine kinase activity is also directed towards other targets of physiological significance. In this work, phosphotyrosine antibodies were used to detect, by Western blots and immunoprecipitation, proteins phosphorylated at tyrosine in fibroblasts either stimulated by growth factors (PDGF and EGF) or transformed by oncogene-coded tyrosine kinases. In stimulated cells the antibodies detected the autophosphorylated receptors, but only trace amounts of other proteins phosphorylated at tyrosine. In fibroblasts transformed by retroviral oncogenes (v-src, v-abl, v-fps or v-fes) proteins other than the corresponding oncogene-coded kinase, were found. A p70 was found to be heavily phosphorylated in fibroblasts transformed by v-src, v-fes and v-fps. A p130 and a p36 were found in cells transformed by v-src and v-abl. A unique p70 was phosphorylated in v-abl-transformed fibroblasts. These proteins were also phosphorylated in vitro in an immunocomplex kinase reaction. This reaction was blocked by the specific kinase inhibitors. These data strongly suggest that tyrosine kinases phosphorylate protein targets other than themselves. These targets are barely detectable in normal cells stimulated by growth factors, where the kinase activity is triggered rapidly and transiently. By contrast, a number of intracellular proteins phosphorylated at tyrosine accumulate in cells transformed by v-onc-coded kinases, endowed with constitutive and non-regulated enzymatic activity.