Immune Complex Kinase Assays Research Articles

Modulation of pp60c‐src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells

High levels of the proto-oncogene product, pp60c-src, have been found in developing and adult neural tissues as well as in certain fully mature cells of the hematopoietic lineage, e.g., platelets and myelomonocytes. Adrenal medullary chromaffin cells exhibit characteristics of both types of cells, i.e., they are derived from the neural crest and carry out exocytosis in response to specific stimuli. Earlier studies have shown that pp60c-src localizes not only to the plasma membrane of chromaffin cells but also to the membranes of chromaffin granules, the secretory vesicles of these cells that store catecholamines and other secretory products. To investigate the possible involvement of pp60c-src in exocytosis, cultured bovine chromaffin cells were analyzed for changes in c-src tyrosine kinase activity in response to stimulation by several secretagogues. Results of in-vitro immune complex kinase assays showed that pp60c-src, derived from cells that had been stimulated for various lengths of time, exhibited decreased auto- and transphosphorylating activities as compared to pp60c-src immunoprecipitated from control cells. The greatest reduction in activity was observed 10 min post-stimulation, while normal levels were regained 2-6 hr after secretagogue treatment. Western immunoblot analysis of the immunoprecipitated pp60c-src revealed that approximately 50% less c-src protein was present in immune complexes prepared 10 min after stimulation as compared to those prepared from mock-stimulated controls, resulting in a specific autophosphorylating activity that was 42-47% of control and little or no reduction in the transphosphorylating specific activity. In experiments in which the rate of secretion of [3H]-norepinephrine from cells preloaded with this compound was compared to the rate of modulation of pp60c-src activity, 50% of the maximal reduction in pp60c-src activity occurred within 2-4 min while 50% maximal release of [3H]-norepinephrine occurred within 1-3 min. Taken together, these results suggest that pp60c-src may play some role (direct or indirect) in the exocytotic process.

Irreversible inhibition of v- src tyrosine kinase activity by herbimycin a and its abrogation by sulfhydryl compounds

Herbimycin A, an antibiotic which reverses Rous sarcoma virus transformation, inhibited irreversibly the auto- and trans-phosphorylation activities of p60v-src in in vitro immune complex kinase assays. The addition of a sulfhydryl compound such as dithiothreitol, 2-mercaptoethanol, glutathione (reduced form) or cysteine abolished the ability of herbimycin A to inactivate p60v-src kinase as well as the ability to reverse transformed cell morphology, whereas the addition of oxidized glutathione, cystine or methionine showed no effect. The sulfhydryl alkylating reagent N-ethylmaleimide also, although less effectively, inactivated p60v-src kinase activity in vitro. These results suggest the likelihood that sulfhydryl groups of p60v-src are involved in the inactivation of v-src tyrosine kinase activity by herbimycin A.

Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity.

We have examined the phosphorylation and the serine/threonine-specific kinase activity of the protooncogene product Raf-1 (formerly c-raf) in response to oncogenic transformation or growth-factor treatment of mouse 3T3 cells. Expression of the membrane-bound oncogene products encoded by v-fms, v-src, v-sis, polyoma virus middle-sized tumor antigen, and Ha-ras increased the apparent molecular weight and phosphorylation of the Raf-1 protein, while expression of the nuclear oncogene and protooncogene products encoded by v-fos and c-myc did not. Changes in electrophoretic mobility and phosphorylation occurred rapidly in response to treatment of cells with platelet-derived growth factor, acidic fibroblast growth factor, epidermal growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate, but not insulin. The phosphorylation of the Raf-1 protein occurred primarily on serine and threonine residues. However, a subpopulation of Raf-1 molecules was phosphorylated on tyrosine residues in cells transformed by v-src or stimulated with platelet-derived growth factor. Transformation by v-src, or treatment with platelet-derived growth factor or phorbol 12-myristate 13-acetate, activated the Raf-1-associated serine/kinase activity as measured in immune-complex kinase assays. These findings suggest that proliferative signals generated at the membrane result in the phosphorylation of the Raf-1 protein and the activation of its serine/threonine kinase activity. Raf-1 activation may thus serve to transduce signals from the membrane to the cytoplasm and perhaps on to the nucleus.

Phosphorylation of a C-SRC-related protein in macrophages activated in vitro with lymphokine

Treatment of proteose peptone elicited peritoneal macrophages from C3H/HeN mice or the macrophage cell line B6MP102 with a T-cell lymphokine preparation induces cytotoxicity for SV3T3 tumor cells. The Triton X-100 (TX-100) insoluble fractions from activated macrophages possessed kinase activity for an endogenous 53 kDa phosphoprotein (pp53) which was markedly greater than extracts from untreated macrophages. Addition of the tyrosine phosphatase inhibitor, Na 3,VO 4 to the cytotoxicity assay also enhanced tumor cell lysis and Na 3VO 4 treated macrophages showed increased phosphorylation of pp53. Moreover, addition of Na 3VO 4 to the cytoskeleton kinase assay enhanced the phosphorylation of pp53 in a dose dependent manner. Pp53 was immunoprecipitated from the in vitro phosphorylated TX-100 insoluble fraction with monoclonal antibody to pp60 v-src. Anti-pp60 v-src also precipitated a 53 and a 60 kDa phosphoprotein from whole cell extracts and from TX-100 cytoskeleton extracts of macrophages phosphorylated as viable intact cells. Addition of a known tyrosine kinase inhibitor, quercetin, to the macrophage cytoskeleton kinase assay inhibited phosphorylation of pp53, and the in vitro phosphorylated pp53 was resistant to 1 N NaOH hydrolysis, indicating phosphorylation of tyrosine residues. Immune complex kinase assays of anti-pp60 c-src precipitated TX-100 insoluble macrophage fractions revealed strong phosphorylation for α-casein which was inhibited by quercetin. These data suggest that macrophage pp53 is a c-src-related gene product that is inducible by stimuli that activate macrophages to cytotoxicity.

C-kit protein, a transmembrane kinase: identification in tissues and characterization.

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).

C-kit Protein, a Transmembrane Kinase: Identification in Tissues and Characterization

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).

Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.

A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.

Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation.

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

Normal Cellular and Transformation-Associated abl Proteins Share Common Sites fOr Protein Kinase C Phosphorylation

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

Identification of an amino terminal domain required for the transforming activity of the Rous sarcoma virus src protein

Transformation of chicken cells by Rous sarcoma virus (RSV) requires the functional expression of the viral src protein, a tyrosine protein kinase, pp60 src . Variants of RSV containing deletions within the amino terminal one-third of the src protein have been identified that exhibit either temperature-sensitive or transformation-defective phenotype when used to infect chicken embryo cells. To define the regions within the amino terminal portion of pp60 src that influence morphological transformation, a series of overlapping deletion mutations in the src gene of Prague RSV (Pr A RSV) were constructed and their biological and biochemical properties were analyzed. Deletions within the src gene which remove amino acid residues 38 to 142 had minimal effects on the ability of the mutant viruses to induce cellular transformation. However, deletions, which impinged upon the region of the src gene encoding residues 142 to 169, inhibited cellular transformation. A variant containing a deletion of amino acid residues 169 to 225, was temperature sensitive for transformation. Structurally altered src proteins recovered from cells infected with transformation-defective variants exhibited a somewhat reduced tyrosine protein kinase activities when assayed in the immune complex kinase assay. Analysis of the in vivo phosphorylation of a pp60 src substrate, the 36-kDa protein, revealed virtually wild-type levels of phosphorylation in cells infected with the transformation-defective mutants. These studies suggest that the region of the Pr A RSV src protein delineated by amino acid residues 142 to 169 is essential for initiation and maintenance of morphological transformation of chicken cells in culture.

Analysis of pp60c‐src tyrosine kinase activity and phosphotyrosyl phosphatase activity in human colon carcinoma and normal human colon mucosal cells

We have compared the level of phosphotyrosyl phosphatase activity in lysates from normal human colon mucosal cells and human colon carcinoma cells and analyzed the effect of incubating these cells with sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatase activity, on the relative abundance of acid-stable phosphotyrosine and on in vitro protein kinase activity of pp60c-src. Additionally, we compared the effect of lysing these cells in buffer containing only nonionic detergents with RIPA buffer, which contains both sodium dodecyl sulfate and deoxycholate, on the in vitro kinase activity of pp60c-src. Our results show that the level of detectable phosphotyrosyl phosphatase activity in lysates derived from normal colon cells and colon carcinoma cells is very similar. Additionally, the abundance of acid-stable phosphotyrosine in these cells cultured in the absence or presence of vanadate is not significantly different. However, incubation of these cells with vanadate significantly stimulates the activity of pp60c-src derived from the normal colon cells in immune-complex kinase assays, while having no detectable effect on the activity of pp60c-src from the colon tumor cells. The in vitro protein kinase activity of pp60c-src derived from RIPA buffer lysates of colon carcinoma cells was found to be elevated five- to sevenfold when compared with pp60c-src from these same cells lysed in buffer containing only Nonidet-P 40 as a detergent. The type of lysis buffer did not effect the activity of pp60c-src from normal colon mucosal cells. These results provide additional evidence that the activity of pp60c-src may be regulated differently in colon carcinoma and normal colon mucosal cells.

The transforming protein of avian reticuloendotheliosis virus is a soluble cytoplasmic protein which is associated with a protein kinase activity

We have identified the product (p57 v- rel ) of the transforming gene, v- rel, of avian reticuloendotheliosis virus (REV-T) using antisera generated against nonoverlapping sequences representing the middle and carboxy-terminal regions of the v- rel protein expressed in Escherichia coli (N. K. Herzog and H. R. Bose, Jr., 1986, Proc. Nat. Acad. Sci. USA 83, 812–816). The amino-terminal region of the v- rel protein was also expressed in E. coli and used to generate antisera. The immunoglobulin-enriched fractions of these antisera were used to determine the subcelluar location of p57 v- rel in REV-T transformed lymphoid cells. Cells were fractionated into nuclear, mitochondrial, microsomal, and cytoplasmic fractions. The majority of p57 v- rel was found in the cytoplasm. Examination of REV-T transformed lymphoid cells labeled with 32P i revealed that the majority of the phosphorylated form of the v- rel protein was also found in the cytoplasm. Indirect immunofluorescence of REV-T transformed cells gave a diffuse cytoplasmic pattern indicating that p57 v- rel was not associated with any discrete cellular organelle. The distribution of p57 v- rel was similar in REV-T transformed lymphoid cells labeled with [ 35S]methionine for short and long periods of time, suggesting that p57 v- rel is a soluble cytoplasmic protein throughout its lifetime. The v- rel protein was phosphorylated when immune complexes precipitated from transformed cells with the immunoglobulin fractions obtained from antisera against the amino-terminal, middle, and carboxyterminal regions of v- rel were incubated with [γ- 32P]ATP and Mn 2+ The phosphorylation of p57 v- rel in the in vitro immune complex kinase assay was inhibited when the immunoglobulin-enriched fraction of these antisera was preincubated with the homologous v- rel fusion proteins. Preincubation with heterologous proteins did not block the phosphorylation of p57 v- rel . These observations suggest that p57 v- rel is associated with a protein kinase activity. Most of the kinase activity was found in the soluble cytoplasmic fraction of transformed cells. The transforming protein encoded by v- rel is a relatively stable protein with a half-life of approximately 7 to 8 hr in transformed lymphoid cells.

Reduced tyrosine kinase specific activity is associated with hypophosphorylation of pp60c-src in cells infected with avian erythroblastosis virus.

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that causes erythroblastosis and sarcomas in chickens and transforms immature erythroid cells and fibroblasts in culture. AEV encodes two oncogenes, v-erbA and v-erbB, whose products are closely related to the thyroxine receptor and the epidermal growth factor receptor, respectively. Since tyrosine protein kinases have been implicated in the process of normal growth signal transduction, we wished to study the possible consequences of the expression of these mutated, growth-regulating receptor genes on the activity of the cellular tyrosine kinase pp60c-src. A continuous cell line from AEV-infected quail embryo fibroblasts was derived that exhibited a typical transformed phenotype and expressed the viral oncogene products, p75gag-erbA and gp66-68erbB. Using an immune-complex kinase assay, we found that the specific activity of pp60c-src in AEV-transformed quail cells was decreased by a factor of 6-30 relative to that found in uninfected quail cells. A concomitant 50-80% reduction of 32Pi incorporation into the pp60c-src protein from radiolabeled, transformed cells was also observed, indicating a relationship between hypophosphorylation and diminished enzyme activity. Partial proteolytic phosphopeptide analysis revealed a decrease in phosphorylation of both serine- and tyrosine-containing peptides, suggesting an activation of specific phosphatases or inhibition of specific kinases in the AEV-transformed quail cells. Similar results were found in pp60c-src precipitated from AEV-transformed chicken and rat cells.

Identification of Molecular Variants of p210bcrabl in Chronic Myelogenous Leukemia

Abstract The aberrant abl protein product of a chronic myelogenous leukemia (CML) blast crisis cell line (K562) and of five Philadelphia chromosome- positive CML patients in blast crisis were analyzed by an immune complex kinase assay using two antipeptide sera generated against the hydrophilic domain of v-abl and a region within the third exon of the breakpoint cluster region (bcr) respectively. Both the anti-abl and anti-bcr sera detected a 210 kd band in extracts derived from K562 cells and from two CML patients with myeloid blast crisis. p210 was detected by the anti-abl but not the anti-bcr sera in three CML patients with myeloid (one patient) and lymphoid (two patients) blast crisis, indicating the absence of bcr exon 3 in this protein. Southern blot analysis on DNA derived from one of the patients in the latter group was consistent with the break on chromosome 22 occurring 5′ to bcr exon 3. Our observations demonstrate that the Philadelphia translocation results in the generation of a chimeric bcr-abl protein with at least two molecular variants, both of which are enzymatically active as protein kinases.

Identification of molecular variants of p210bcr-abl in chronic myelogenous leukemia

The aberrant abl protein product of a chronic myelogenous leukemia (CML) blast crisis cell line (K562) and of five Philadelphia chromosome-positive CML patients in blast crisis were analyzed by an immune complex kinase assay using two antipeptide sera generated against the hydrophilic domain of v-abl and a region within the third exon of the breakpoint cluster region (bcr) respectively. Both the anti-abl and anti-bcr sera detected a 210 kd band in extracts derived from K562 cells and from two CML patients with myeloid blast crisis. p210 was detected by the anti-abl but not the anti-bcr sera in three CML patients with myeloid (one patient) and lymphoid (two patients) blast crisis, indicating the absence of bcr exon 3 in this protein. Southern blot analysis on DNA derived from one of the patients in the latter group was consistent with the break on chromosome 22 occurring 5' to bcr exon 3. Our observations demonstrate that the Philadelphia translocation results in the generation of a chimeric bcr-abl protein with at least two molecular variants, both of which are enzymatically active as protein kinases.

Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells.

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.

Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells.

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.

In vivo effect of sodium orthovanadate on pp60c-src kinase.

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

In Vivo Effect of Sodium Orthovanadate on pp60c-src Kinase

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

Cellular proteins homologous to the viral yes gene product.

We raised antibodies in rabbits against the amino-terminal portion of the viral yes protein produced in bacteria with the use of an expression vector based on the lac operon. The anti-yes serum thus obtained precipitated P90gag-yes from Yamaguchi 73 virus-transformed chicken embryo fibroblasts, and this immunoprecipitation was blocked by the purified antigen. The anti-yes serum did not recognize viral src, fps, or fgr proteins. Affinity-purified anti-yes immunoglobulin G (IgG) precipitated two proteins of 59 and 62 kilodaltons from lysates of normal chicken embryo fibroblasts. Two-dimensional tryptic peptide mapping showed that these proteins are closely related to P90gag-yes and that they are different from pp60c-src. Similar to P90gag-yes, the 59- and 62-kilodalton proteins were phosphorylated exclusively on tyrosine in an in vitro kinase reaction, whereas in vivo they were phosphorylated on serine and, to a lesser extent, on tyrosine as well. Expression of the 59- and 62-kilodalton proteins, determined by the immune complex kinase assay, was relatively high in brain, retina, kidney, and liver. The presence in normal chicken embryo fibroblasts and in chicken kidney of two transcripts, 3.7 and 3.9 kilobases in length, that hybridize with a yes-specific DNA probe, as well as the two proteins recognized by anti-yes IgG, suggests either differential splicing of cellular yes gene transcripts or the existence of another yes-related gene.