Biomedical subjects

S J Parsons

Publications and source records attributed to S J Parsons.

At least 73 records · Page 4Linked to original sources

PubMed1990

A 42-kD tyrosine kinase substrate linked to chromaffin cell secretion exhibits an associated MAP kinase activity and is highly related to a 42-kD mitogen-stimulated protein in fibroblasts.

The localization of the protein tyrosine kinase pp60c-src to the plasma membrane and to the membrane of secretory vesicles in neurally derived bovine chromaffin cells has suggested that tyrosine phosphorylations may be associated with the process of secretion. In the present study we have identified two cytosolic proteins of approximately 42 and 45 kD that become phosphorylated on tyrosine in response to secretagogue treatment. Phosphorylation of these proteins reached a maximum (3 min after stimulation) before maximum catecholamine release was observed (5-10 min after stimulation). Both secretion and tyrosine phosphorylation of p42 and p45 required extracellular Ca2+. Tyrosine-phosphorylated proteins of similar Mr have previously been identified in 3T3-L1 adipocytes stimulated with insulin (MAP kinase; Ray, L. B., and T. W. Sturgill. 1987. Proc. Natl. Acad. Sci. USA. 84:1502-1506) and in avian and rodent fibroblasts stimulated with a variety of mitogenic agents (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273; Nakamura, K. D., R. Martinez, and M. J. Weber. 1983. Mol. Cell. Biol. 3:380-390). Comparisons of the secretion-associated 42-kD protein of chromaffin cells with the 42-kD protein of Swiss 3T3 fibroblasts and 3T3-L1 adipocytes provide evidence that these three proteins are highly related. This evidence includes comigration during one-dimensional SDS-PAGE, cochromatography using ion exchange and hydrophobic matrices, similar isoelectric points, identical cyanogen-bromide peptide maps, and cochromatography of MAP kinase activity with the tyrosine-phosphorylated form of pp42. This protein(s), which appears to be activated in a variety of cell types, may serve a common function, perhaps in signal transduction involving a cascade of kinases.

Adrenal Gland Neoplasms↗

PubMed1990

Enhanced EGF mitogenic response is associated with enhanced tyrosine phosphorylation of specific cellular proteins in fibroblasts overexpressing c-src.

In previous studies examining the role of pp60c-src in cellular proliferation, we demonstrated that overexpression of wild type (wt) but not mutated c-src in C3H10T1/2 murine embryo fibroblasts resulted in a 2 to 5 fold enhancement of DNA synthesis in response to epidermal growth factor (EGF). Using phosphotyrosine antiserum to probe Western immunoblots of whole cell lysates, we determined in the present study that proteins migrating at approximately 170 (EGF receptor), 125, 100, 75, 63 and 57 kDa exhibited EGF-dependent tyrosine phosphorylations that were approximately 3 fold higher in the wt c-src overexpressors than in control cells, while proteins of 125, 100 and 75 kDa displayed basal, unstimulated levels that were approximately 2 fold higher. Pp60c-src-associated hyperphosphorylation of all but the 63 and 57 kDa proteins was reduced or ablated in cells overproducing structurally-altered forms of c-src. Furthermore, in contrast to control cells, wt c-src overexpressors retained EGF-induced tyrosine phosphorylations on the 125, 100 and 75 kDa proteins for up to 9 h of EGF treatment. These results illuminate potential substrates for pp60c-src and suggest that overproduced pp60c-src potentiates the EGF mitogenic response by increasing the extent and duration of tyrosine phosphorylations on proteins involved in EGF mitogenic signalling.

Animals↗

PubMed1989

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.

Adrenal Medulla↗

PubMed1989

pp60c-src tyrosine kinase, myristylation, and modulatory domains are required for enhanced mitogenic responsiveness to epidermal growth factor seen in cells overexpressing c-src.

In previous studies examining the potential role of pp60c-src in cellular proliferation, we demonstrated that C3H10T1/2 murine embryo fibroblasts overexpressing transfected chicken genomic c-src displayed an epidermal growth factor (EGF)-induced mitogenic response which was 200 to 500% of the response exhibited by parental control cells (Luttrell et al., Mol. Cell. Biol. 8:497-501, 1988). In order to examine specific structural and functional requirements for pp60c-src in this event, 10T1/2 cells were transfected with chicken c-src genes encoding pp60c-src deficient in tyrosine kinase activity (pm430), myristylation, (pm2A), or a domain hypothesized to modulate the interaction with substrates or regulatory components (dl155). Neomycin-resistant clonal cell lines overexpressing each of the mutated c-src genes were assayed for EGF mitogenic responsiveness by measuring [3H]thymidine incorporation into acid-precipitable material or into labeled nuclei. The results were compared with those obtained with lines overexpressing the cDNA form of wild-type (wt) c-src or control cells transfected with the neomycin resistance gene only. As previously described for cells overexpressing wt genomic c-src (Luttrell et al., 1988), clones overexpressing wt cDNA c-src also exhibited enhanced EGF mitogenic responses ranging from approximately 300 to 400% of the control cell response. In contrast, clones overexpressing unmyristylated, modulation-defective, or kinase-deficient c-src not only failed to support an augmented response to EGF but also exhibited EGF responses lower than that of the control cells. Furthermore, there were no significant differences in the mitogenic responses to 10% fetal calf serum among any of the cells tested. These results indicate that pp60(c-scr) can potentiate mitogenic signaling generated by EGF but not all growth factors. This potentiation requires the utilization of pp60(c-scr) myristylation, and modulatory and tyrosine kinase domains and can me mediated by cDNA-encoded as well as by genome-encoded wt pp60(c-scr).

Animals↗

PubMed1989

Insulin and phorbol ester induce distinct phosphorylations of pp60c-src in the BC3H-1 murine myocyte cell line.

Insulin and phorbol esters have been shown to produce similar, non-additive metabolic effects in BC3H-1 murine myocytes. Recently, it has been demonstrated that insulin stimulation of these cells increases production of diacylglycerol, a known activator of protein kinase C (PK-C). To determine if insulin stimulation results in the activation of PK-C, we have examined the effects of insulin and the tumor promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the phosphorylation of a known PK-C substrate in vivo, the cellular proto-oncogene product, pp60c-src. Differentiated BC3H-1 monocytes showed an approximate twofold elevation in the [32P] content of pp60c-src following stimulation with insulin or TPA for 20 min, with no detectable change in the level of immunoprecipitable c-src protein. The enhanced phosphorylation in response to each agent localized to serine residues in the amino terminal 16 kD staphylococcal V8 proteolytic fragment. Tryptic phosphopeptide analysis revealed that TPA stimulation resulted in an approximate 18-fold increase in phosphorylation of the serine 12-containing tryptic fragment. Insulin stimulation, however, resulted in an approximate 10-fold increase in phosphorylation of the serine 17-containing tryptic fragment with little or no accompanying increase in serine 12 phosphorylation. In cells exposed to high concentrations of TPA for 16 h to deplete PK-C activity, insulin, but not TPA, stimulated phosphorylation of pp60c-src. These data suggest that insulin and phorbol ester induce phosphorylation of pp60c-src by distinct protein kinases.

Animals↗

PubMed1988

Augmented mitogenic responsiveness to epidermal growth factor in murine fibroblasts that overexpress pp60c-src.

C3H10T1/2 murine fibroblasts overexpressing chicken pp60c-src showed a two- to fivefold enhanced incorporation of [3H]thymidine into DNA in response to epidermal growth factor (EGF) relative to that of the parent line. No difference in growth characteristics, number and affinity of EGF receptors, or hormone potency was attributable to c-src overexpression. These results suggest that pp60c-src may interact with the mitogenic signal transduction pathway of EGF in some event distal to hormone binding.

Animals↗

PubMed1988

Activation of pp60c-src tyrosine kinase specific activity in tumor-derived Syrian hamster embryo cells.

Tumor-derived Syrian hamster embryo (SHE) cell lines, induced in vitro by treatment with chemical carcinogens, contained increased levels of pp60c-src kinase activity compared to preneoplastic parental cell lines and normal SHE cells. The increased kinase activity did not result from an increase in the pp60c-src content of the SHE cell lines, but represented a 4-11 fold increase in pp60c-src kinase specific activity. Both the extent of phosphorylation and the velocity of pp60c-src phosphotransferase activity were increased in the tumor-derived cell lines. SHE cell lines producing chicken pp60c-src were isolated following co-transfection with plasmids bearing the chicken c-src and neoR genes. Chicken pp60c-src expressed in an asbestos-transformed, tumor-derived cell line showed an approximate 3-fold activation of tyrosine kinase activity compared to chicken pp60c-src expressed in the preneoplastic cell line. We suggest that these results indicate that activation of pp60c-src is mediated by trans-acting cellular factors present in the tumor-derived cells. Analysis of pp60c-src in normal SHE cells, preneoplastic cell lines and tumor-derived cell lines showed no alteration in the phosphorylation of tyr-527 or tyr-416, two tyrosine residues whose phosphorylation states have been associated with modulation of kinase activity. These studies indicate that the neoplastic progression of cells may be accompanied by the activation of proto-oncogene products, such as the pp60c-src tyrosine kinase, by mechanisms that may not directly involve genetic alteration of the proto-oncogene DNA sequence.

Animals↗

PubMed1987

Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients.

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.

Amino Acids↗

PubMed1987

Identification of chromaffin granule-binding proteins. Relationship of the chromobindins to calelectrin, synhibin, and the tyrosine kinase substrates p35 and p36.

At least 23 soluble proteins (chromobindins) bind to chromaffin granule membranes in the presence of Ca2+. In order to further the identification of the chromobindins and to determine the roles they may play in exocytosis or other aspects of chromaffin cell biology, several of these proteins were compared to other known membrane-binding proteins. Chromobindin 4 was identified as a 32-kDa protein called calelectrin or endonexin. Immunologically related proteins were detected in bovine brain and human platelets. Chromobindin 20 was identified as a 67-kDa variant of calelectrin and was found to have the activities of the synexin inhibitory protein, synhibin. Chromobindin 8 was identified as p36, a substrate for the tyrosine-specific kinase, pp60v-src. Chromobindin 8 was also demonstrated to undergo phosphorylation predominantly on alkali-sensitive sites during stimulation of the chromaffin cell with 20 microM nicotine. Chromobindin 6 was identified as p35, a substrate for the tyrosine kinase activity associated with the epidermal growth factor receptor. Chromobindin 9, which is known to be a substrate for protein kinase C (Ca2+/phospholipid-dependent enzyme), was found to be immunologically related to p35 and may be a precursor of chromobindin 6. The identification of these proteins from the chromaffin system may be useful in the characterization of similar, complex groups of membrane-binding proteins that have been observed in other systems.

Amino Acids↗

PubMed1987

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.

Alpharetrovirus↗

PubMed1987

Inhibition of the tyrosine kinase activity of v-src, v-fgr, and v-yes gene products by a monoclonal antibody which binds both amino and carboxy peptide fragments of pp60v-src.

A monoclonal antibody, R2D2, raised to the src gene product of Rous sarcoma virus was found to inhibit the tyrosine protein kinase activity of pp60v-src in autophosphorylation reactions and in reactions involving exogenously added substrates, such as casein and histone. R2D2 also inhibited the enzymatic activity of two related viral transforming proteins, pp70gag-fgr and pp90gag-yes. The inhibitory ability of R2D2 was dependent upon immunoglobulin concentration and could be demonstrated in both immune complexes formed directly with R2D2 and preformed immune complexes to which R2D2 was added. Binding sites in both the amino-terminal 110 amino acid residues and the carboxy-terminal 240 amino acids of pp60v-src were identified for R2D2. These results indicate that at least part of the epitope recognized by R2D2 resides within a region of the src protein which is required for protein kinase activity. The localization of the R2D2 epitope to the amino- as well as to the carboxy-terminal portions of pp60v-src, together with results of studies analyzing the relative binding efficiencies of R2D2 to the intact protein and to V-8 proteolytic fragments of pp60v-src, are consistent with the view that the R2D2 epitope is conformational in nature and that it is assembled from residues contained within both N-terminal and C-terminal regions of the molecule.

Antibodies, Monoclonal↗

PubMed1986

p60c-src activity detected in the chromaffin granule membrane.

Using monoclonal antibodies specific for p60c-src we have detected high levels of this kinase in adrenal medullary chromaffin tissue and in highly purified chromaffin granule (secretory vesicle) membranes. An immune complex kinase assay was applied to fractions of adrenal medullary tissue resolved on sucrose density gradients. Thirty-seven per cent of the total tissue p60c-src activity was found in association with chromaffin granule or granule membrane markers. Localization of a significant fraction of total cellular p60c-src activity to this secretory vesicle membrane suggests that the kinase may function in the regulation of neurotransmitter release.

Adrenal Cortex↗

PubMed1986

Localization of conserved and nonconserved epitopes within the Rous sarcoma virus-encoded src protein.

We investigated the location of binding sites of pp60src-specific monoclonal antibodies by immunoprecipitating a panel of structurally altered src proteins. Two families of antibodies which recognized epitopes mapping to either amino acid residues 28 to 38 or 92 to 128 were identified. The highly conserved nature of the epitope defined by residues 92 to 128 suggests that it may represent an important functional region of the cellular src protein.

Antibodies, Viral↗

PubMed1986

Structural and functional motifs of the Rous sarcoma virus src protein.

Site-directed mutagenesis techniques have been utilized to define important structural and functional domains within the RSV src gene product, pp60src. Deletion mutations within the amino terminal one-half of the src gene which impinge upon a region of the src protein delineated by amino acid residues 143 to 169 yielded transformation defective viruses. Src proteins encoded by such RSV mutants exhibited diminished tyrosine protein kinase activity in vitro and only slightly reduced levels of in vivo tyrosine protein kinase activity. We speculate that these structurally altered proteins are defective for target protein recognition. Point mutations and linker insertion mutations within the putative catalytic domain of pp60src served to block the transforming activity of mutant viruses. Mutant viruses encode src proteins that exhibited substantially reduced levels of tyrosine protein kinase activity both in vitro and in vivo.

Amino Acid Sequence↗

PubMed1984

Monoclonal antibodies specific for the virion polypeptide, p27, of avian retroviruses.

The major component of the core structure of avian sarcoma leukosis viruses is a 27 kD molecular weight polypeptide, p27. Spleen cells from mice immunized with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV) were fused with mouse myeloma cells (SP2/0), and hybridoma cell lines producing monoclonal antibodies to p27 were isolated. The monoclonal antibodies were all of the IgG1 subclass with kappa light chains. These antibodies immunoprecipitated p27 and its precursor proteins from extracts of RSV-transformed cells. Reciprocal competitive binding experiments defined five nonoverlapping antigenic determinants within p27. The monoclonal antibodies also immunoprecipitated the transforming protein, p110gag-myc, from avian myelocytomatosis virus transformed cells. Their usefulness in studies of virion maturation and viral oncogenesis is discussed.

Alpharetrovirus↗

PubMed1984

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin.

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.

Animals↗

PubMed1983

Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src.

Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src. A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein.

Animals↗

PubMed1979

Immune response to the src gene product in mice bearing tumors induced by injection of avian sarcoma virus-transformed mouse cells.

A single subcutaneous injection of 10(7) live cells of the highly tumorigenic avian sarcoma virus (Schmidt-Ruppin strain, subgroup D)-transformed BALB/c line into BALB/c mice resulted in the production of an antiserum specific for the avian sarcoma virus gene product pp60src. All sera taken from mice 3 weeks after injection of tumor cells contained antibodies to pp60src. Immunoprecipitation experiments showed that all sera precipitated pp60src from Schmidt-Ruppin-infected chicken cells, but only a portion of these sera precipitated pp60src from chicken cells infected with other strains of avian sarcoma virus, i.e., Prague and Bratislava-77. Analysis of the cross-reactivity patterns of these antisera demonstrated a minimum of three to four antigenic determinants on pp60src. The findings reported here should facilitate the production of monoclonal antibodies to pp60src, which in turn will provide highly specific probes for further investigations into the structure and function of this protein.

Animals↗