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.     

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.     

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.     

Structure and phosphorylation of the Fujinami sarcoma virus gene product

The Fujinami avian sarcoma virus (FSV) transforming gene product, P140, is a fusion protein which contains both gag-related and FSV-specific methionine-containing tryptic peptides. The virion protease p15 cleaved p140 into two fragments: an N-terminal 33K fragment which contained all but one of the gag-related tryptic peptides and a C-terminal 120K fragment which contained all of the FSV-specific tryptic peptides. The 33K gag-related fragment from P140 phosphorylated in FSV-transformed cells contained only phosphoserine, whereas the 120K C-terminal FSV-specific fragments contained both phosphoserine and phosphotyrosine. P140 isolated from cells infected at the nonpermissive temperature with an isolate of FSV which is temperature sensitive for transformation had a normally phosphorylated 33K fragment, but a hypophosphorylated 120K fragment deficient in both phosphotyrosine and phosphoserine. When P140 was immunoprecipitated from cells and phosphorylated in vitro at tyrosine residues in the immune complex kinase reaction, only the FSV-specific fragment was labeled. These data define the structure of FSV P140 and locate the phosphorylated amino acids within the two regions of the polypeptide.     

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.     

Temperature-Sensitive Mutants of Fujinami Sarcoma Virus: Tumorigenicity and Reversible Phosphorylation of the Transforming p140 Protein

Several clones of Fujinami sarcoma virus (FSV) isolated from a laboratory stock or from mutagenized virus were temperature sensitive (ts) in transformation of cells in culture. When shifted from the permissive (37°C) to the nonpermissive (41.5°C) temperature, the cellular phenotype reverted to normal within 2 h, but it required about 48 h at 37°C to revert back to the transformed morphology. A temperature-resistant (tr) FSV clone was isolated from a tumor of an animal. All ts mutants were tumorigenic in animals but induced tumors only after latent periods of 12 to 25 days, compared to 5 to 6 days with tr virus. The ts lesions of the FSV mutants affected 90% of the phosphorylation of the nonstructural, gag-related 140,000-kilodalton phosphoprotein coded by FSV (p140), but did not affect virus replication or the synthesis of p140. Upon shifting from the permissive to the nonpermissive temperature, p140 was 90% dephosphorylated with an approximate ³²P half-life of 20 min. When shifted back to the permissive temperature, the preexisting p140 was rephosphorylated in the absence of protein synthesis within a 90-min test period. Likewise, most of the phosphate of fully phosphorylated p140 was exchanged at the permissive temperature within 30 to 90 min even when protein synthesis was inhibited. However, the protein structure of p140 had a half-life of 5 h at both temperatures. These results prove p140 to be a substrate of reversible phosphorylation. Superinfection and transformation of ts FSV-infected cells maintained at the nonpermissive temperature with acute leukemia virus MC29 failed to phosphorylate p140. It would follow that in vivo phosphorylation of ts p140 is controlled by an FSV-specific mechanism and is a prerequisite, not a consequence, of transformation. p140 of ts FSV recovered from cells maintained at 41.5°C with anti-gag serum was over 10 times less phosphorylated by associated kinase than the same protein recovered from cells at 37°C if assayed in vitro at 20°C. This kinase activity associated with or dissociated from p140 with a half-life of less than 30 min during temperature shifts of ts FSV-infected cells. However, p140 recovered from ts FSV-infected cells maintained at 37°C was phosphorylated by associated kinase in vitro not only at 20°C but also, and essentially at the same level, at 41.5°C. This suggests that the kinase associated with the immunocomplex of p140 of ts FSV is not temperature sensitive. p140 translated in vitro from ts and tr FSV RNA lacked kinase activity. We conclude that a fully phosphorylated p140 is necessary for the maintenance of transformation by FSV. This is consistent with the notion that other highly oncogenic viruses also code for nonstructural phosphoproteins with probable transforming function. A model which postulates that p140 is a substrate of reversible phosphorylation and that the lesion of the ts FSV clones described herein affects association of p140 with a cellular kinase rather than a hypothetical intrinsic kinase activity of the protein is most compatible with our data.     

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.     

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.     

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.     

gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

Cells transformed by the McDonough strain of feline sarcoma virus express at their surface a v-fms-specific transmembrane glycoprotein designated gp140v-fms. By labeling with 32Pi, gp140v-fms was shown to be phosphorylated 30-fold more in serine residues than were the cytosolic v-fms polypeptides gp180gag-fms and gp120v-fms. By using the phosphotyrosine phosphatase-specific inhibitor sodium orthovanadate, an additional tyrosine phosphorylation was observed in vivo, again involving predominantly gp140v-fms. In vitro studies showed that the v-fms proteins were phosphorylated by protein kinase C in a calcium- and phosphatidylserine-dependent manner.     

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.     

A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ

A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.     

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.     

A 56,000 Mr phosphoseryl protein in PC12 cell lysates strongly associates with protein-A sepharose beads and was observed in immune complex kinase assays for PP60c-src

Using an immune complex kinase assay to measure pp60c-src kinase activity, we have identified a 56,000 M_r protein (p56) from PC12 cell lysates that co-purified with pp60c-src by strong association with protein-A sepharose beads. The p56 protein was strongly phosphorylated on serine but no tyrosine or threonine phosphorylation was evident. However, pp60c-src was strongly phosphorylated on tyrosine, weakly phosphorylated on serine with no observed threonine phosphorylation. P56 was not a proteolytic breakdown product of pp60c-src, since it was neither tyrosine phosphorylated nor was it recognized by anti-src antibody. P56 was also not recognised by other antibodies to 56kD signalling molecules such as p56lck. The identity of p56 awaits further investigation but its appearance in immunoprecipitates of pp60c-src using protein-A sepharose beads is of interest but complicates the interpretation of results from immune complex kinase assays in PC12 cells.     

Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.     

Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis.

Fujinami sarcoma virus (FSV) encodes a 140,000-dalton transforming protein, P140, which contains gag- and fps-specific sequences. The cellular localization of this protein was examined by fractionation of [35S]methionine-labeled, FSV-infected chicken embryo fibroblasts. In homogenates of cells infected by wild-type, temperature-resistant FSV prepared in either hypotonic or isotonic buffer, 60 to 80% of the P140 was particulate. Isopycnic separation on discontinuous sucrose gradients indicated that the majority of the particulate P140 was present in a light membrane fraction enriched for plasma membranes. Much of the particulate P140 could be solubilized by the addition of 0.6 M salt to a postnuclear supernatant, suggesting that P140 is not an integral membrane protein. Particulate P140 may be associated with membranes either directly as a peripheral membrane protein or indirectly via cytoskeletal elements. In cells infected by mutants of FSV temperature sensitive for cellular transformation, most of the P140 is particulate at the permissive temperature, whereas most is soluble at the nonpermissive temperature; this change in distribution is not a secondary consequence of the change in cellular phenotype, since it also occurs in nonconditionally transformed cells doubly infected with temperature-sensitive FSV and wild-type Rous sarcoma virus. The movement of P140 from the particulate to the soluble fraction occurs rapidly when cells infected by temperature-sensitive FSV are shifted from the permissive to the nonpermissive temperature. Furthermore, P140 moves from the soluble to the particulate fraction, although somewhat more slowly, when cells are shifted from the nonpermissive to the permissive temperature. These observations suggest that the association of P140 with plasma membranes or the cytoskeleton may play a role in transformation by FSV.     

Human immunodeficiency virus matrix tyrosine phosphorylation: characterization of the kinase and its substrate requirements.

During virus assembly, a subset of human immunodeficiency virus (HIV) matrix (MA) molecules is phosphorylated on C-terminal tyrosine. This modification facilitates infection of nondividing cells by allowing for the recruitment of the karyophilic MA into the viral core and preintegration complex. MA tyrosine phosphorylation is accomplished by a cellular protein kinase which is incorporated into virions. In this study, we have investigated the nature of this enzyme as well as the determinants of MA necessary for its phosphorylation. Employing an in vitro kinase assay, we found that the MA tyrosine kinase activity is present in various cultured cell lines including CEM and SupT1 T-lymphoid cells, Namalwa B cells, 293 and CV-1 kidney fibroblasts, and P4 HeLa cells. In addition, it could be detected in platelets, macrophages, and activated peripheral blood lymphocytes (PBLs) but not in erythrocytes and resting PBLs isolated from human blood. Subcellular localization of the kinase activity by cell fractionation demonstrated that it is enriched in cellular membranes. In HIV type 2 (HIV-2) particles, the MA tyrosine kinase is associated with the inner leaflet of the viral membrane, while the tyrosine-phosphorylated MA is localized to the core. Individual mutations of each of the last eight residues immediately upstream of the C-terminal tyrosine (Y132) of HIV-1 MA did not prevent Y132 phosphorylation, suggesting that the kinase does not require a highly specific sequence adjacent to the C-terminal tyrosine. Confirming this, we found that the MA of murine leukemia virus, the sequence of which is only moderately homologous to that of HIV-1 and HIV-2 MA, is also C-terminally tyrosine phosphorylated.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus.

The gag gene-related, nonstructural proteins of three avian acute leukemia viruses (namely, myelocytomatosis viruses MC29 and CMII and avian erythroblastosis virus) and of avian Fujinami sarcoma virus (FSV) isolated by immunoprecipitation from cellular lysates with anti-gag serum were shown to be phosphoproteins in vivo. The specific 32P radioactivity of the nonstructural proteins of MC29, CMII, and FSV was significantly higher than that of helper viral, intracellular gag proteins. Two of these proteins, i.e., the 140,000-dalton FSV and the 110,000-dalton MC29 proteins, were also phosphorylated in vitro by a kinase activity associated with immunocomplexes. This kinase activity is either separated from these proteins or inactivated by incubation of cellular lysates with normal serum followed by adsorption to staphylococcal protein A or sedimentation at 100,000 x g or both. It remains to be resolved whether the 110,000-dalton MC29 and 140,000-dalton FV proteins, in addition to being substrates for phosphorylation, also have intrinsic kinase activity.     

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.