Cellular localization of c-fps gene product NCP98.

We compared the intracellular location of the product of the c-fps proto-oncogene, NCP98, with that of its viral homolog P140, the transforming protein of Fujinami sarcoma virus. Using the technique of biochemical subcellular fractionation, we determined that 60 to 90% of NCP98 and its associated kinase activity are in the soluble fraction of a chicken myeloblast cell line. This fractionation behavior differs from that of P140, which is found predominantly in the particulate fraction, both in Fujinami sarcoma virus-infected chicken embryo fibroblasts and in Fujinami sarcoma virus-infected myeloblasts. The fractionation behavior of NCP98 is, however, similar to that of the P140 encoded by a temperature-sensitive strain of Fujinami sarcoma virus in infected cells grown at the nonpermissive temperature. The absence of gag sequences from NCP98 is not responsible for the difference in fractionation behavior: the v-fps transforming protein of strain F36, P91, which lacks gag sequences, is also predominantly particulate. These results indicate that association with cellular structural components correlates with the transforming activity of proteins containing fps sequences.     

Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family.     

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.     

Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells.

We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140gag-fps was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from P140gag-fps, indicating that P140gag-fps has no detectable PI kinase activity.     

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.     

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.     

Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.     

A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation

Cells infected by one strain of Fujinami sarcoma virus (FSV) are transformed at 38 degrees C but are phenotypically normal at 41.5 degrees C. FSV encodes a 140,000 molecular weight protein (P140) with gag gene-related and FSV-specific peptide sequences. At 41.5 degrees C, P140 is weakly phosphorylated at serine residues, and is inactive in the immune complex protein kinase assay. At 38 degrees C, P140 is highly phosphorylated, contains phosphotyrosine in addition to phosphoserine, and in the immune complex kinase assay becomes phosphorylated at three tyrosine residues. Phosphorylation of cellular polypeptides at tyrosine residues in FSV-infected cells is also temperature-sensitive. These observations indicate that P140 is the transforming protein of FSV and that protein phosphorylation at tyrosine residues is involved in transformation by this virus.     

Mutants of Fujinami sarcoma virus which are temperature sensitive for cellular transformation and protein kinase activity.

Two temperature-sensitive mutants of Fujinami sarcoma virus were isolated and characterized. Cells infected with the mutants were temperature sensitive in focus formation, colony formation, increased sugar uptake, and synthesis of plasminogen activator. The changes between transformed and nontransformed states of cultures were completely reversible by shifting the temperature. A Fujinami sarcoma virus-specific protein of 130,000 daltons, p130, was synthesized in mutant-infected cells regardless of the temperature, but the immunoprecipitates of p130 from extracts of infected cells were active in protein kinase only when cells had been incubated at the permissive temperature. These results appear to indicate that p130 is the transforming protein of Fujinami sarcoma virus, and that its protein kinase activity plays a crucial role in cell transformation by this virus.     

Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus

Fujinami sarcoma virus (FSV), a newly characterized avian sarcoma virus, produces a protein of 140,000 daltons (p140) in infected cells. p140 is the product of a fused gene consisting of a part of the gag gene of avian retrovirus and FSV-unique sequences which are not related to the src sequences of Rous sarcoma virus. In vivo, p140 was found to be phosphorylated at both serine and tyrosine residues. Immunoprecipitates of p140 with antiserum against gag gene-coded proteins had a cyclic nucleotide-independent protein kinase activity which phosphorylated p140 itself, rabbit IgG of the immune complex and alpha-casein, an externally added soluble protein substrate. The phosphorylation was specific to tyrosine of the substrate proteins. p140 was phosphorylated in vitro at the same two tyrosine residues that were phosphorylated in vivo. The phosphate transferred to tyrosine residues of p140 forms a stable bond: it does not turn over during the kinase reaction, and the 32P-phosphate of p140 labeled in vitro or in vivo is not transferred to alpha-casein. FSV-p140 differs from p60src, the transforming protein of Rous sarcoma virus, in its marked preference of Mn2+ to Mg2+ ions, and in its inability to use GTP instead of ATP as the donor of gamma-phosphate.     

Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.     

Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.     

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.     

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.     

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.     

The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes.

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.     

In vivo tyrosine phosphorylations of the abelson virus transforming protein are absent in its normal cellular homolog

The transforming gene product of the Abelson murine leukemia virus (A-MuLV) is a phosphoprotein encoded by combined viral and cellular sequences. Previous work has shown the existence of a serologically crossreactive normal cellular phosphoprotein called NCP150. We have utilized two-dimensional phosphopeptide mapping and phosphoamino acid analysis to compare the structures of NCP150 and wild-type and mutant forms of the A-MuLV protein labeled in vivo with ³²P-orthophosphate. This analysis demonstrated clear homology between NCP150 and wild-type A-MuLV protein, but a number of phosphorylation differences were seen. Among them, two specific tyrosine phosphorylations present in all transformation-competent Abelson proteins were not observed in NCP150. No other phophotyrosine-containing peptides were detected. In addition, transformation-defective mutants isolated from either the P120 or P160 wild-type strain lack phophotyrosine-containing peptides. Double-infection studies with such transformation-defective and transformation-competent AMuLV strains show that Abelson viral proteins may be substrates for their own tyrosine-specific kinase activity in vivo. These observations suggest that the phosphotyrosine kinase activity of the abl region may be controlled, and may function, differently in its viral and cellular forms.     

Specific inhibition of tyrosine kinase activity by an antibody to the v-ros oncogene product.

Antibodies present in two peritoneal exudates of rats bearing abdominal tumors induced by UR2-transformed rat cells were characterized. The ability to immunoprecipitate p68gag-ros and to inhibit the protein and phospholipid kinase activities of this protein was investigated. One of the exudates specifically inhibited tyrosyl phosphorylation by p68gag-ros but not the activity of other known tyrosyl kinases, such as p150gag-fps of UR1 avian sarcoma virus, p60src, and the insulin receptor. It precipitated p68gag-ros but not Pr76 or other gag-related proteins from UR2-infected cells. Phosphorylation of phosphatidylinositol was not affected by this exudate, suggesting that this activity is not intrinsic to p68gag-ros. Another exudate precipitated p68gag-ros but not gag-related proteins from UR2-infected cells or p140gag-fps from Fujinami sarcoma virus-infected cells. These results demonstrated that the antibodies in these exudates recognized epitopes present in the ros portion of the fused protein p68gag-ros, but only one of the two exudates inhibited the intrinsic tyrosyl kinase of p68gag-ros.     

Inhibition of tyrosine protein kinases by halomethyl ketones

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.     

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.