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.     

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.     

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.     

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.     

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.     

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.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.     

Phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase of synthetic peptide analogs of a site phosphorylated in histone H2B

Analogs of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B were used to assess the substrate specificity of cGMP-dependent protein kinase. cGMP-dependent kinase phosphorylated the oligopeptide Arg-Lys-Arg-Ser32-Arg-Lys-Glu with favorable kinetic parameters as compared to those for cAMP-dependent kinase (Glass, D. B., and Krebs, E. G. (1979) J. Biol. Chem. 254, 9728-9738). The contribution of each amino acid to the ability of the peptide to be phosphorylated by cGMP-dependent or cAMP-dependent kinase was studied by replacement of individual residues and evaluation of the kinetic constants of the substituted peptides. Peptides containing acetylated lysine residues or nitroarginine residues were poor substrates for both kinases. Substitution of either arginine 29 or lysine 30 with alanine increased the Km values and decreased the Vmax values for both kinases. Substitution of lysine 34 with alanine increased the Vmax values for both kinases but did not affect the Km values for either enzyme. Substitution of the phosphorylatable serine with a threonine residue greatly depressed the Vmax for both kinases. Peptides in which arginine 31 or arginine 33 were replaced by an alanine residue revealed several apparent differences in the specificity requirements between cGMP-dependent and cAMP-dependent kinases.     

Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.     

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.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.     

Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527).

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.     

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.     

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.     

Activated Src tyrosine kinase phosphorylates Tyr-457 of bovine GTPase-activating protein (GAP) in vitro and the corresponding residue of rat GAP in vivo.

GTPase-activating protein (GAP) is a key regulator of the cellular Ras protein, which is implicated in oncogenic signal transduction pathways downstream of the viral Src (v-Src) kinase. Previous studies demonstrated that v-Src induces tyrosine phosphorylation of GAP, suggesting that GAP may provide a biochemical link between v-Src and Ras signaling pathways. To determine the precise residues in GAP phosphorylated by Src kinases, we used a baculovirus/insect cell expression system for investigating in vitro phosphorylation of GAP. Phosphopeptide mapping analysis revealed that v-Src and normal cellular Src (c-Src) phosphorylate tyrosine residues in bovine GAP at one major site and one minor site in vitro. Significantly, the major site of GAP phosphorylation in vitro is also the major site of in vivo tyrosine phosphorylation of GAP in rat fibroblasts transformed by v-Src. Analyses of GAP deletion mutants and TrpE-GAP fusion proteins established that Tyr-457 of bovine GAP (and the corresponding residue of rat and human GAP) is the major site of tyrosine phosphorylation. Our results demonstrate that the v-Src kinase induces phosphorylation of the same tyrosine residue of GAP in vitro and in vivo, suggesting that GAP is a direct substrate of activated Src kinases in vivo. Because epidermal growth factor receptor phosphorylates the equivalent tyrosine residue in human GAP (Tyr-460), these findings are consistent with the hypothesis that specific phosphorylation of GAP at this site may have a physiologically important role in regulating mitogenic Ras signaling pathways.     

Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1.

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.     

Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.     

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.