Src family protein tyrosine kinases: cooperating with growth factor and adhesion signaling pathways.

The Src family of protein tyrosine kinases functionally interacts with several receptor and nonreceptor protein tyrosine kinases. Recent developments show that Src family kinases may cooperate with the epidermal growth factor and platelet-derived growth factor receptors and the integrin-linked focal adhesion kinase to diversify signals that regulate growth and cell movement.

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.

Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190-RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K- cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190.

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.

c-Src modulates ErbB2 and ErbB3 heterocomplex formation and function.

Overexpression and/or gene amplification of c-Src and members of the epidermal growth factor receptor (EGFR/ErbB) family have been implicated in the pathogenesis of breast cancer. Although members of the EGFR family are known to form heterocomplexes with one another, c-Src has also been shown to physically interact with members of this family in breast cancer cell lines and tumors. This paper investigates the role of c-Src in modulating the physical and functional interaction between ErbB2 and ErbB3, two family members that preferentially associate with one another and together exhibit high oncogenic potential. We show that overexpressed wild-type c-Src enhances heterocomplex formation of ErbB2 and ErbB3 that results in increased basal and/or heregulin-induced activation of receptors, and their downstream intracellular effectors. Expression of a kinase-inactive form of c-Src (K(-) c-Src) or pharmacological inhibition of c-Src by PP2 negatively affects these events. Furthermore, cellular motility and anchorage-independent growth promoted by the ErbB2/ErbB3 heterocomplex are dependent upon c-Src, as demonstrated by the effects of K(-) c-Src overexpression or treatment with PP2. In contrast to previous studies that defined a role for c-Src downstream of ErbB2/ErbB3, the current work suggests an upstream mechanism, whereby c-Src enhances ErbB2/ErbB3 signaling and biological functions by positively modulating the association between ErbB2 and ErbB3.

Neurotensin stimulates mitogenesis of prostate cancer cells through a novel c-Src/Stat5b pathway.

Neuroendocrine (NE)-like cells are hypothesized to contribute to the progression of prostate cancer by producing factors that enhance the growth, survival or metastatic capabilities of surrounding tumor cells. Many of the factors known to be secreted by NE-like cells, such as neurotensin (NT), parathyroid hormone-related peptide, serotonin, bombesin, etc., are agonists for G-protein-coupled receptors, but the signaling pathways activated by these agonists in prostate tumor cells are not fully defined. Identification of such pathways could provide insights into novel methods of treating late-stage disease. Using conditioned culture medium (CM) from LNCaP-derived NE-like cells (as a source of these agonists) or NT (a prototypical component of CM) to treat PC3 cells, we found that the epidermal growth factor (EGF) receptor (EGFR) was transactivated and that such activation was required for maximal PC3 cell mitogenesis, as measured by 5-bromo-2'-deoxy-uridine incorporation or cell number. NT also induced a time-dependent increase in EGFR Tyr(845) phosphorylation and phosphorylation of c-Src and signal transducer and activator of transcription 5b (Stat5b) (a downstream effector of Tyr(845)), events that were blocked by specific inhibition of c-Src (which mediates Tyr(845) phosphorylation of EGFR) or of EGFR. Introduction of mutant forms of EGFR (Tyr(845)) or Stat5b in PC3 cells, or treatment with selective, catalytic inhibitors of EGFR, c-Src and matrix metalloproteinases (MMPs) resulted in the loss of NT-induced stimulation of DNA synthesis, relative to wild-type controls. These data indicate that the mitogenic effect of NT on prostate cancer cells requires transactivation of the EGFR by MMPs and a novel downstream pathway involving c-Src, phosphorylation of EGFR Tyr(845) and activation of Stat5b.

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.

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.

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).

Interleukin-6- and cyclic AMP-mediated signaling potentiates neuroendocrine differentiation of LNCaP prostate tumor cells.

Neuroendocrine (NE) differentiation in prostatic adenocarcinomas has been reported to be an early marker for development of androgen independence. Secretion of mitogenic peptides from nondividing NE cells is thought to contribute to a more aggressive disease by promoting the proliferation of surrounding tumor cells. We undertook studies to determine whether the prostate cancer cell line LNCaP could be induced to acquire NE characteristics by treatment with agents that are found in the complex environment in which progression of prostate cancer towards androgen independence occurs. We found that cotreatment of LNCaP cells with agents that signal through cyclic AMP-dependent protein kinase (PKA), such as epinephrine and forskolin, and with the cytokine interleukin-6 (IL-6) promoted the acquisition of an NE morphological phenotype above that seen with single agents. Convergent IL-6 and PKA signaling also resulted in potentiated mitogen-activated protein kinase (MAPK) activation without affecting the level of signal transducer and activator of transcription or PKA activation observed with these agents alone. Cotreatment with epinephrine and IL-6 synergistically increased c-fos transcription as well as transcription from the beta4 nicotinic acetylcholine receptor subunit promoter. Potentiated transcription from these elements was shown to be dependent on the MAPK pathway. Most importantly, cotreatment with PKA activators and IL-6 resulted in increased secretion of mitogenic neuropeptides. These results indicate that PKA and IL-6 signaling participates in gene transcriptional changes that reflect acquisition of an NE phenotype by LNCaP cells and suggest that similar signaling mechanisms, particularly at sites of metastasis, may be responsible for the increased NE content of many advanced prostate carcinomas.

Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP-p120 RasGAP interaction: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation.

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.

A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.

Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible: implications for prostate cancer progression.

Neuroendocrine (NE) cells occur as scattered foci within prostatic adenocarcinoma, similar to their distribution within ductal epithelial cells of the normal prostate. However, the density of NE cells is often greater in prostate carcinomas than in normal tissue, and the frequency of NE cells correlates with tumor grade, loss of androgen sensitivity, autocrine/paracrine activity, and poor prognosis. Although NE cells are nonmitotic, proliferating cells are found in direct proximity to them, suggesting that NE cells provide paracrine stimuli for surrounding carcinoma cells. In vitro, differentiation of the LNCaP and PC3M prostatic tumor cell lines to a NE phenotype can be induced by dibutyryl cyclic AMP (cAMP), suggesting that physiological agents that increase intracellular concentrations of cAMP might regulate NE differentiation in vivo. Indeed, we demonstrate in this report that LNCaP cells acquire NE characteristics in response to treatment with physiological and pharmacological agents that elevate intracellular cAMP, agents such as epinephrine, isoproterenol, forskolin, and dibutyryl cAMP. The androgen-independent LNCaP-derived cell line C4-2 also responded to these agents, indicating that cells representing later stages of tumor progression are also capable of differentiation. The NE phenotype in this study was monitored by the appearance of dense core granules in the cytoplasm, the extension of neuron-like processes, loss of mitogenic activity, and expression of the NE markers neuron-specific enolase, parathyroid hormone-related peptide, neurotensin, serotonin, and chromogranin A. However, contrary to previous reports, we observed rapid loss of the NE phenotype in both LNCaP and C4-2 cells upon withdrawal of inducing agents. Withdrawal also resulted in a rapid, dramatic increase in tyrosine kinase and mitogen-activated protein kinase activities, suggesting that activation of these intracellular signaling enzymes may be important for reentry into the cell cycle. Together, these results indicate that chronic cAMP-mediated signaling is required to block proliferation of prostate tumor cells and to induce NE differentiation.

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.

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.

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.

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.

Increased levels of p21ras-GTP and enhanced DNA synthesis accompany elevated tyrosyl phosphorylation of GAP-associated proteins, p190 and p62, in c-src overexpressors.

While examining the role of pp60c-src in cellular proliferation, we found that overexpression of c-src in C3H10T1/2 murine fibroblasts results in an augmented mitogenic response to epidermal growth factor (EGF) [Luttrell, D.K., Luttrell, L.M. & Parsons, S.J. (1988). Mol. Cell. Biol., 8, 497-501; Wilson, L.K., Luttrell, D.K., Parsons, S.J. (1989). Mol. Cell. Biol., 9, 1536-1544] and enhanced tyrosyl phosphorylation of specific cellular proteins [Wilson, L.K. & Parsons, S.J. (1990). 5, 1471-1480]. Here we identify two of these proteins as the GAP (GTPase-activating protein of p21ras)-associated proteins, p190 and p62. Evidence is presented to support the notion that, in 10T1/2 fibroblasts, p190 is a preferred substrate of pp60c-src, while p62 is preferentially phosphorylated by the EGF receptor. First, the phosphotyrosine content of p190 in quiescent cells is three- to fivefold higher in c-src overexpressors than in control cells and is not altered by growth factor treatment. In contrast, tyrosyl phosphorylation of p62 is undetectable in quiescent cells and transiently observable upon EGF addition. Second, the phosphotyrosine content of p190 in cells overexpressing defective pp60c-src is reduced in comparison with wild-type (wt) c-src overexpressors, while that of p62 is significantly less affected. Further studies revealed that tyrosyl phosphorylation of p190 and p62 is not required for GAP complex formation, as equal amounts of p190 and p62 proteins could be detected in GAP complexes from wt and variant c-src overexpressors both before and after EGF stimulation. However, analysis of GTP-bound p21ras revealed higher basal and EGF-stimulated levels in c-src overexpressors than in control cells. Taken together, these results suggest that one mechanism by which pp60c-src may contribute to early events in the EGF-induced mitogenic pathway in 10T1/2 fibroblasts is by increasing the level of GAP-associated p190 and p62 tyrosyl phosphorylation, which in turn results in higher levels of p21ras-GTP.

Src family kinases and HER2 interactions in human breast cancer cell growth and survival.

Evidence from murine fibroblast models and human breast cancer cells indicates that c-Src and human EGF receptor (HER1) synergize to enhance neoplastic growth of mammary epithelial cells. To investigate whether interactions between c-Src and other HER family members may also play a role in breast tumor progression, we characterized 13 human breast carcinoma cell lines and 13 tumor samples for expression of HER family members and c-Src and examined a subset of the cell lines for Src-dependent, heregulin (HRG)-augmented, anchorage-dependent and independent growth. By immunoblotting, we found that all cell lines overexpressed one or more HER family member, and 60% overexpressed c-Src. Seventy-five per cent of the tumor tissues overexpressed HER2, while 64% overexpressed c-Src. Colony formation in soft agar was enhanced by HRG in three of five cell lines tested, a response that correlated with the presence of a c-Src/HER2 heterocomplex. This result suggests that HRG may act through both HER2 and c-Src to facilitate anchorage-independent growth. In contrast, HRG had little effect on anchorage-dependent growth in any of the cell lines tested. PP1, a Src family kinase inhibitor, reduced or ablated HRG-dependent and independent soft agar growth or anchorage dependent growth, and triggered apoptosis in all cell lines tested. The apoptotic effect of PP1 could be partially or completely reversed by HRG, depending on the cell line. These results suggest that while Src family kinases may cooperate with HRG to promote the survival and growth of human breast tumor cells, they also function independently of HER2/HRG in these processes.

Identification and characterization of a cytoskeleton-associated, epidermal growth factor sensitive pp60c-src substrate.

In studies aimed at identifying and characterizing pp60c-src substrates that participate in the enhanced mitogenic response to epidermal growth factor (EGF) observed in murine C3H10T1/2 fibroblasts overexpressing c-src, we have identified a 75-kDa protein (p75) whose properties are consistent with those expected of such a substrate. We present evidence to show that p75 is immunologically related to a recently described, cytoskeleton-associated, pp60v-src substrate [Wu et al. (1991). Mol. Cell. Biol., 11, 5113-5124), and that its phosphotyrosine content is increased cooperatively by c-src overexpression and EGF stimulation. p75 is rapidly (within 2 min) phosphorylated on tyrosine upon EGF treatment and undergoes a second, prolonged phase of tyrosyl phosphorylation from 7 to 21 h after EGF addition, suggesting that tyrosyl phosphorylation of p75 is important for late as well as early events following EGF receptor activation. Enhanced tyrosyl phosphorylation of p75 is also seen when cells overexpressing c-src are treated with platelet-derived growth factor (PDGF), but significantly less phosphorylation is observed with insulin and fibroblast growth factor (FGF). Both basal and EGF-induced tyrosyl phosphorylation of p75 are reduced in cells overexpressing mutated forms of c-src (unmyristylated, or kinase deficient) as compared with wild-type c-src overexpressers, indicating the dependence of the enhanced tyrosyl phosphorylation on membrane-associated, enzymatically active pp60c-src. In cellular fractionation experiments p75 partitions with the cytosol, while immunofluorescence studies reveal a striking colocalization with pp60c-src at the plasma membrane and in the perinuclear region. Partial co-staining of p75 and actin occurs at the cell's periphery. These data provide evidence for p75 being a direct substrate of pp60c-src. The possible role of p75 in the enhanced response to EGF seen in c-src overexpressers is discussed.

Overexpression of pp60c-src is associated with altered regulation of adenylyl cyclase.

The ability of activators of the beta-adrenergic receptor to elevate intracellular cAMP levels in murine fibroblasts is enhanced upon overexpression of avian c-src [Bushman et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 7462-7466]. To investigate the molecular basis for this effect, we prepared particulate fractions from control and pp60c-src overexpressing C3H10T1/2 fibroblasts and assessed the relative abilities of several activators of the beta-adrenergic receptor-Gs-adenylyl cyclase (AC) signal transduction pathway to stimulate the enzymatic response. Two- to three-fold increases in both the sensitivity and maximum responsiveness of AC to the beta-adrenergic agonist isoproterenol were consistently observed in fractions prepared from the c-src overexpressing cells. Interestingly, the AC response to two agents believed to act directly at the level of the G protein were either enhanced (NaF) or unaffected (GTP gamma S) by c-src overexpression. Finally, overexpression of c-src was associated with a reduced ability of both Mn2+ and forskolin to activate AC directly. These results suggest that overexpression of wild type c-src may affect two distinct steps in the regulation of AC exerting a positive effect at the level of Gs activation and a negative effect on AC itself. As no differences in the relative number or affinity of beta-adrenergic receptors, or in the level of AC, Gs alpha or G beta, were detected between control cells and those overexpressing c-src, we propose that pp60c-src overexpression results in a modification of one or more components in this signal transduction pathway.