Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk.

A recombinant baculovirus was constructed for the production of the serine-specific protein kinase, pp90rsk (where rsk is ribosomal S6 kinase), in insect cells. The Xenopus pp90rsk expressed in the infected cells had nearly undetectable enzyme activity in contrast to the same enzyme coproduced with the v-src oncogene product pp60v-src. The transforming gene product pp60v-src very effectively activated pp90rsk, whereas the products of c-src and the myristoylation-minus nontransforming virus NY315 were markedly less effective. Only a fraction of the total pp90rsk population was activated, and it could be partially separated from unactivated protein by ion-exchange chromatography. When compared to the unactivated form, the activated enzyme displayed about a 4000-fold increase in the capacity to phosphorylate the ribosomal protein S6. The enhanced enzymatic activity appeared to be due to phosphorylation of pp90rsk.     

pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions.

Expression of the Rous sarcoma virus-encoded oncoprotein, pp60v-src, subverts the normal regulation of cell growth, which results in oncogenic transformation. This process requires the intrinsic protein-tyrosine kinase activity of pp60v-src and is associated with an increase in tyrosine phosphorylation of a number of cellular proteins, candidate substrates for pp60v-src. We report here the isolation of a cDNA encoding a protein, pp125, that is a major phosphotyrosine-containing protein in untransformed chicken embryo cells and exhibits an increase in phosphotyrosine in pp60v-src-transformed chicken embryo cells. This cDNA encodes a cytoplasmic protein-tyrosine kinase which, based upon its predicted amino acid sequence and structure, is the prototype for an additional family of protein-tyrosine kinases. Immunofluorescence localization experiments show that pp125 is localized to focal adhesions; hence, we suggest the name focal adhesion kinase.     

Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src).

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp60v-src and pp60-c-src are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v-src and pp60c-src. We have therefore characterized the sites of tyrosine phosphorylation in the two proteins. Tyrosine phosphorylation of pp60v-src in infected cells occurs mainly (if not entirely) at residue 419 in the deduced amino acid sequence of the protein. Surrounding this residue is the sequence Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg. This peptide is distinguished by the fact that three out of the four amino acids that precede the phosphorylated tyrosine are acidic in nature. These results define what may prove to be a widely used site for tyrosine phosphorylation in the regulation of cellular function. The same site was phosphorylated when partially purified pp60v-src was used in a phosphotransfer reaction in vitro. The results with pp60c-src were more complex. The site of tyrosine phosphorylation in vitro appeared to be the same as that found in pp60v-src. By contrast, phosphorylation of pp60c-src in vivo apparently occurred at a different, and currently unidentified, tyrosine residue. It is therefore possible that pp60v-src and pp60c-src respond differently to regulatory influences in the intact cell.     

The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor.

We have observed a modification of the cellular protein kinase pp60c-src, elicited in murine 3T3 fibroblasts by platelet-derived growth factor (PDGF). The modification occurred rapidly after addition of PDGF to the culture medium and was first detected as a reduction in the electrophoretic mobility of a portion of the pp60c-src molecules. A similarly modified form of the viral homologue pp60v-src occurs in vivo in the absence of stimulation by PDGF. The occurrence of modified forms of both pp60c-src and pp60v-src was associated with a novel phosphorylation at tyrosine in the amino-terminal domains of the proteins. The time-course and dose-response for this modification of pp60c-src paralleled PDGF-induced increases in phosphorylation of pp36, a major cellular substrate for several tyrosine-specific protein kinases. In parallel experiments, treatment of cells with PDGF increased the kinase activity of pp60c-src in an immunocomplex assay. These results suggest pp60c-src may play a role in the mitogenic response to PDGF.     

Characterization of avian and viral p60src proteins expressed in yeast.

Avian and viral p60src proteins were expressed from a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. Both the viral and cellular src proteins produced in yeast cells were myristoylated at their amino termini, as is the case for src proteins expressed in chicken embryo fibroblasts. The viral src protein produced in yeast autophosphorylated at tyrosine-416 in vivo and had approximately the same level of in vitro kinase activity as p60v-src expressed in Rous sarcoma virus-transformed cells. Unlike p60c-src expressed in chicken cells, which is phosphorylated on tyrosine in vivo almost exclusively at tyrosine-527, p60c-src expressed in yeast was phosphorylated 2.5-3 times more at tyrosine-416 than at tyrosine-527. The specific activity of the p60c-src produced in yeast was 2.5-5.0 times higher than that of p60c-src overexpressed from a retroviral vector in chicken cells, implicating the altered state of in vivo phosphorylation in modulation of the in vitro kinase activity. The expression of p60v-src substantially slowed down the growth of the yeast cells, suggesting that phosphorylation of yeast proteins essential for cell growth may have interfered with their proper functioning.     

Transit of pp60v-src to the plasma membrane.

The protein kinase (pp60v-src) encoded by the transforming gene (v-src) of Rous sarcoma virus is synthesized on free polyribosomes and then translocated to the plasma membrane of infected cells. Neither the mechanism of the translocation nor the physiological significance of the membrane localization has been elucidated. We have explored these problems by pursuing previous observations of a complex between pp60v-src and two cellular proteins with molecular weights of 50,000 and 89,000. We found the complex located entirely in the cytoplasm, where it appears to form immediately after the synthesis of pp60v-src. While in the complex, pp60v-src has little detectable kinase activity and is phosphorylated predominantly on serine. After transfer from the complex to the plasma membrane, pp60v-src becomes phosphorylated on tyrosine as well as serine and acquires kinase activity. Under restrictive conditions, temperature-sensitive pp60v-src is produced in normal quantities, but translocation to the plasma membrane is diminished. As an apparent consequence, the cytoplasmic complex accumulates to abnormal abundance. Alternatively, temperature-sensitive pp60v-src that has been synthesized and translocated to the plasma membrane under permissive conditions appears to be released from the membrane and returns to the cytoplasmic complex when the infected cells are shifted to the restrictive temperature. We conclude that the cytoplasmic complex may be the vehicle by which pp60v-src reaches the plasma membrane. It is possible that other proteins may follow a similar route to the membrane. Binding to plasma membrane appears to be a discrete step in the biogenesis of pp60v-src and may be essential to the function of the protein.     

A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: positive in vivo regulation by sterol.

The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp60v-src were examined in a sterol auxotroph of Saccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v-src shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, approximately equal to 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control.     

Expression of v-src in a murine T-cell hybridoma results in constitutive T-cell receptor phosphorylation and interleukin 2 production.

Ligand binding to the T-cell antigen receptor results in phosphatidylinositol hydrolysis and the resultant activation of protein kinase C, as well as the activation of a receptor-coupled protein-tyrosine kinase. As a model for tyrosine kinase activation in T cells, we used retroviral gene transfer to express the v-src oncogene in an antigen-specific murine T-cell hybridoma. Clones that expressed v-src mRNA demonstrated constitutive tyrosine phosphorylation of several cellular substrates, including the zeta chain of the T-cell receptor, and constitutive interleukin 2 production. Thus, expression of a constitutively active protein-tyrosine kinase such as pp60v-src appears to be sufficient to induce the expression of at least one gene critical to the process of T-cell activation.     

Phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide of pp60c-src.

The major site of tyrosine phosphorylation of the transforming protein of Rous sarcoma virus, pp60v-src (tyrosine-416), is different from the major site of tyrosine phosphorylation of its nontransforming normal cellular counterpart, pp60c-src. We have shown that antibodies against a synthetic peptide modeled on the carboxyl-terminal 13 residues of pp60c-src specifically immunoprecipitate the major phosphotyrosine tryptic peptide of pp60c-src from both chicken and rat fibroblasts. These experiments localize the major site of tyrosine phosphorylation to one or more of the three tyrosine residues in the carboxyl-terminal tryptic peptide at positions 511, 519, and 527 of the amino acid sequence of chicken pp60c-src. Tyrosines-519 and -527 are in the carboxyl-terminal 19-amino acid segment of pp60c-src that is deleted and replaced by an unrelated sequence in pp60v-src. It is possible that phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide may be involved in the normal regulation of pp60c-src. The absence of this phosphorylation site in pp60v-src may, in part, contribute to its oncogenic properties.     

Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src.

Protein phosphatase 1, one of four major protein phosphatases involved in cellular regulation, was phosphorylated in vitro by pp60v-src, the transforming gene product of Rous sarcoma virus. Phosphorylation was accompanied by a loss of protein phosphatase activity. The inactivation of protein phosphatase 1 was time-dependent and the extent of inactivation correlated closely with the stoichiometry of phosphorylation. Under optimal conditions, 0.34 +/- 0.01 mol of phosphate were incorporated per mol of protein phosphatase and the activity of the enzyme was decreased by 39 +/- 2%. The inactivation required the presence of both MgATP and pp60v-src. There was no loss of activity when adenosine 5'-[beta gamma-imido]triphosphate was used in place of ATP. Phosphorylation of protein phosphatase 1 occurred exclusively on tyrosine residues and was blocked by specific antibodies to pp60v-src. During preincubation of pp60v-src at 41 degrees C, its protein kinase activity towards casein was lost rapidly. The ability of pp60v-src to phosphorylate and inactivate protein phosphatase 1 declined in parallel with the loss of casein kinase activity. Limited chymotryptic digestion of 32P-labeled protein phosphatase 1 (Mr 37,000) resulted in its quantitative conversion to a Mr 33,000 species. Conversion to this species was accompanied by the loss of 32P-labeling and by reactivation of the protein phosphatase. When various concentrations of chymotrypsin were used in the digestion, there was a close correlation between conversion to the Mr 33,000 species and the restoration of protein phosphatase activity. pp60v-src was unable to phosphorylate or inactivate a partially proteolyzed species of protein phosphatase 1 (Mr 33,000/34,000).     

Evidence from two transformed cell lines that the phosphorylations of peptide tyrosine and phosphatidylinositol are catalyzed by different proteins.

Two transformed rodent cell lines (RS-1 and LSTRA) were studied in vitro to determine if their major protein tyrosine kinases catalyzed the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or diacylglycerol. RS-1 cells, transformed by Rous sarcoma virus, contain high levels of pp60src; LSTRA cells, transformed by Moloney murine leukemia virus, contain a tyrosine kinase (pp56) that is the product of an unknown cellular gene. Rates of phosphorylation of peptide tyrosine were elevated more than 20-fold in RS-1 and LSTRA particulate fractions compared to fractions from suitable control cells (N2 and YAC-1), but there was not a proportional increase in rates of phosphorylation of PtdIns, PtdIns4P, or diacylglycerol. Heat (34 degrees C) completely inactivated the LSTRA tyrosine kinase, while it enhanced the phosphorylation of PtdIns and PtdIns4P and had no effect on the phosphorylation of diacylglycerol. PtdIns4P inhibited the phosphorylation of PtdIns but had no effect on tyrosine kinase activity. An antibody, raised against a peptide with a sequence homologous to the autophosphorylation site of pp60src, immunoprecipitated tyrosine kinase activity from RS-1 and LSTRA extracts but had no effect on PtdIns kinase or PtdIns4P kinase activity. These results provide evidence that the phosphorylations of tyrosine and PtdIns are catalyzed by different proteins. An additional observation was that a monoclonal antibody that binds to pp60src and pp56 removed PtdIns kinase as well as tyrosine kinase activity from RS-1 and LSTRA particulate extracts. This antibody also removed PtdIns kinase from N2 and YAC-1 extracts, in which tyrosine kinase activity was low or undetectable. Thus, the anti-pp60src monoclonal antibody may recognize the PtdIns kinase in addition to pp60src and pp56.     

GTPase-activating protein interactions with the viral and cellular Src kinases.

GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v-src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src.     

Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.

Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.     

Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11.

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.     

Transforming gene product of Rous sarcoma virus phosphorylates tyrosine.

The protein kinase activity associated with pp60src, the transforming protein of Rous sarcoma virus, was found to phosphorylate tyrosine when assayed in an immunoprecipitate. Despite the fact that a protein kinase with this activity has not been described before, several observations suggest that pp60src also phosphorylates tyrosine in vivo. First, chicken cells transformed by Rous sarcoma virus contain as much as 8-fold more phosphotyrosine than do uninfected cells. Second, phosphotyrosine is present in pp60src itself, at one of the two sites of phosphorylation. Third, phosphotyrosine is present in the 50,000-dalton phosphoprotein that coprecipitates with pp60src extracted from transformed chicken cells. We infer from these observations that pp60src is a novel protein kinase and that the modification of proteins via the phosphorylation of tyrosine is essential to the malignant transformation of cells by Rous sarcoma virus. pp60sarc, the closely related cellular homologue of viral pp60src, is present in all vertebrate cells. This normal cellular protein, obtained from both chicken and human cells, also phosphorylated tyrosine when assayed in an immunoprecipitate. This is additional evidence of the functional similarity of these structurally related proteins and demonstrates that all uninfected vertebrate cells contain at least one protein kinase that phosphorylates tyrosine.     

Etoposide-mediated deregulation of the G2M checkpoint in myeloid leukaemic cell lines results in loss of cell survival

The K562 leukaemic cell line expresses an inherent survival signal due to the antiapoptotic properties of Bcr-abl, which is, in part, mediated by prolonging the G2M checkpoint and allowing DNA repair mechanisms to operate post genotoxic insult. Arrest of the cell cycle is mediated by retaining an inactivating state of phosphorylation of cyclin-dependent kinase 1 (Cdk1) on tyrosine 15. Our data confirmed that cell survival in K562 was promoted by cell cycle arrest at G2M in response to the genotoxin etoposide. There was no predicted cell cycle arrest in Bcr-abl-positive derivative cell lines of K562 that did not survive the same genotoxic insult but, paradoxically, Cdk1 tyrosine phosphorylation was enhanced to a higher extent compared with the parental cell line where arrest of the cell cycle was observed. To ascertain that this was not an anomaly of the derivative lines, HL60 cells were treated with concentrations of etoposide that induced arrest of the cell cycle or apoptosis. Only HL60 cells that subsequently underwent apoptosis elicited the same effect of increased Cdk1 tyrosine phosphorylation. It is proposed that the augmented tyrosine phosphorylation status of Cdk1 is associated with the abolition of cell survival, in addition to the previously reported induction of cell cycle arrest in myeloid cell lines.     

pp60c-src kinase expression in brain of adult rats in relation to age.

Although there is evidence of alterations in brain protein phosphorylation patterns with age, it is not known if the protein kinases that phosphorylate only at tyrosine residues are involved in these changes. For this reason, we examined the age-related expression of pp60c-src, a tyrosine protein kinase enriched in neural tissues, in whole brain of adult Fischer-344 rats. The pp60c-src kinase activity was immunoprecipitated using a monoclonal antibody and the incorporation of [32P] from radiolabeled ATP into an exogenous substrate (casein) measured. The results showed that there was a substantial amount of pp60c-src kinase activity in brain of the adult animals ranging in age from 4 to 23 months and that it was not significantly different among these groups. Also, immunoprecipitates obtained under conditions of monoclonal antibody excess and utilized for immunoblot analysis indicated that the relative levels of the pp60c-src protein were unchanged in the same animals. These results suggest that, at the whole brain level, the pp60c-src kinase has a stable turnover and that a high amount of activity is biologically important in brain of adult rats through early senescence.     

Identification of the tyrosine protein kinase from LSTRA cells by use of site-specific antibodies.

The lymphoma cell line LSTRA contains an elevated level of tyrosine protein kinase activity. It has been suggested that this elevated level of activity is due to the presence of a phosphoprotein with a molecular weight of 56,000 (pp56, formerly referred to as a 58,000-dalton protein). This paper describes the preparation of antibodies against pp56 through the use of a synthetic peptide that contains the sequence around the site of tyrosine phosphorylation in pp56, which is identical to the phosphorylation site in pp60src. These antipeptide antibodies specifically immunoprecipitated 32P-labeled pp56 from detergent extracts of LSTRA cells. In immunoblotting experiments, pp56 was the major antigen detected in the particulate fraction from LSTRA cells by the antipeptide antibodies. The antibodies were also used to show that the level of pp56 is greatly elevated in LSTRA cells. Incubation of the detergent extract of the particulate fraction from LSTRA cells with the antipeptide antibodies resulted in inhibition of most of the LSTRA cell tyrosine protein kinase activity. These results indicate that pp56 is the tyrosine protein kinase whose activity is elevated in LSTRA cells. This enzyme may be a member of the large family of protein kinases involved in the regulation of cell growth.     

Epidermal growth factor stimulates the phosphorylation of synthetic tyrosine-containing peptides by A431 cell membranes.

A431 cell membranes phosphorylate a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gly) in which residues 2--12 correspond to the sequence of the reported site of tyrosine phosphorylation in pp60src. Epidermal growth factor stimulates the phosphorylation of this peptide 2-fold over basal levels in a dose-dependent fashion. Phosphorylation is linear for approximately 3 min at 30 degrees C and occurs on the tyrosine residue. Kinetic analysis of the phosphorylation reaction indicates that epidermal growth factor increases the average Vmax from 3.8 to 7.5 nmol/min per mg and slightly decreases the average Km from 0.53 mM to 0.28 mM. A number of other peptides analogous to this tridecapeptide are also phosphorylated by A431 membranes. The data suggest that peptides with sequences similar to the site of tyrosine phosphorylation in pp60src are preferred substrates for the kinase in these membranes. Thus, the epidermal growth factor-stimulated protein kinase has the potential to interact with and phosphorylate pp60src. However, the A431 membranes also phosphorylate a tyrosine-containing peptide of totally unrelated sequence, suggesting that the kinase possesses a broad specificity for peptide phosphorylation that may not reflect its specificity with protein substrates.