Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

We have shown previously that pp60c-src is a substrate for protein kinase C in vivo and that the target of protein kinase C phosphorylation in mammalian pp60c-src is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2 alpha) lead to the phosphorylation of pp60c-src at serine 12. In addition to stimulating serine 12 phosphorylation in pp60c-src, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.     

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.     

Regulation of pp60c-src expression in rat and mouse fibroblasts by an inducible antisense gene: effects on serum regulation of growth and polyoma virus middle T function.

Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum.     

Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.     

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.     

Novel serine phosphorylation occurs in the fibroblast form of pp60c-src from Y79 retinoblastoma cells.

Two-dimensional tryptic peptide analysis showed that pp60c-src from the human retinoblastoma cell line Y79 gave a unique phosphopeptide, which was not found in human fibroblast RT59. There was no significant difference in the extent of phosphorylation of other peptides between the two cell lines. Only phosphoserine was detected in this phosphopeptide. Both the fibroblast form and the neuronal form of pp60c-src from Y79 cells had this unique peptide phosphorylated to the same extent. The phosphorylation site was inferred to be serine 97 by comparing the tryptic map and the arginyl-endopeptidase map. The specific protein kinase activity of pp60c-src from Y79 cells was nearly equal to that of RT59 pp60c-src. This unique serine phosphorylation in the fibroblast form was discussed in relation to the oncogenic change of Y79 cells.     

Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect.

Madin-Darby canine kidney (MDCK) cells are highly differentiated and have retained the morphogenetic properties necessary to form polarized, multicellular epithelial structures (cysts) in vitro that resemble epithelial tissues in vivo. We introduced the c-src gene into MDCK cells to elevate the level of the plasma membrane-associated cellular tyrosine kinase, pp60c-src, to levels two- to ninefold higher than that expressed in parent MDCK cells. Our results revealed a highly discriminatory biological action of pp60c-src on the morphogenetic properties of MDCK cells. Elevated expression of pp60c-src conferred on MDCK cells the ability to undergo dramatic changes of cell shape that includes the formation of long cell processes (100 to 200 microns), never observed in control MDCK cells. The morphogenesis of multicellular epithelial cysts was altered by elevated levels of pp60c-src and led to predictable distortions of their three-dimensional architecture. However, these cells established morphologically normal cell polarity, formed adhesive epithelial cell-cell contacts indistinguishable from those of control MDCK cells, and exhibited neither focus-forming ability or anchorage-independent growth potential. Finally, we showed that MDCK cells expressing elevated levels of pp60c-src exhibit increased phosphorylation of a more limited number of phosphotyrosine-containing proteins than MDCK cells expressing pp60v-src. We suggest that a natural function of pp60c-src is to regulate the morphogenetic properties which determine the shape of differentiated cells and multicellular structures.     

Altered phosphorylation and activation of pp60c-src during fibroblast mitosis

At least half the pp60c-src in NIH 3T3-derived c-src overexpresser cells in modified by novel threonine and, possibly, serine phosphorylation within its amino 16 kd region during mitosis. At the same time, the tryptic phosphopeptide containing Ser 17, the site of cyclic AMP-dependent phosphorylation, is either modified or dephosphorylated. While the amount of pp60c-src is not significantly altered, the in vitro-specific kinase activity of modified pp60c-src is enhanced 4- to 7-fold. Modified pp60c-src has the same tyrosine-containing tryptic phosphopeptides as pp60c-src from unsynchronized cells, indicating that activation is independent of Tyr 416/Tyr 527 phosphorylation. Electrophoretic mobility retardations indicated that endogenous pp60c-src and pp60v-src are similarly modified during mitosis. The modifications and enhanced activity disappear near the time of cell division. These results suggest that pp60c-src is regulated by and, in turn, may regulate mitosis-specific events in fibroblasts.     

pp60c-src in human melanocytes and melanoma cells exhibits elevated specific activity and reduced tyrosine 530 phosphorylation compared to human fibroblast pp60c-src.

Elevated levels of pp60c-src tyrosine kinase activity have been implicated in both tumorigenesis and cell differentiation. We have found a 2- to 4-fold elevation in pp60c-src specific activity in certain human melanoma cell lines compared to human foreskin fibroblasts. This activation of pp60c-src did not appear to be related to melanoma tumor progression, because when normal human epidermal melanocytes were examined, it was found that they contained pp60c-src having a 7-fold elevation in specific activity compared to pp60c-src from human fibroblasts. It was determined that pp60c-src from melanocytes was not the neuronal form, pp60c-src+. Melanocyte pp60c-src exhibited a reduced level of phosphorylation on its carboxyl-terminal regulatory site, tyrosine 530, which might be responsible for its elevated specific activity. These results suggest that, in melanocytes, regulation of tyrosine 530 phosphorylation-dephosphorylation favors activation of pp60c-src. This activation may be involved in the growth, differentiation, or function of human melanocytes.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.     

Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon. Cell invasion occurs via bacterium-directed phagocytosis, a process requiring polymerization of actin at the site of bacterial entry. We show that invasion of HeLa cells by S.flexneri induces tyrosine phosphorylation of cortactin, a host cell protein previously identified as a cytoskeleton-associated protein tyrosine kinase (PTK) substrate for the proto-oncoprotein pp60c-src. Immunolocalization experiments indicate that cortactin is recruited to submembranous actin filaments formed during bacterial entry. In particular, cortactin is highly enriched in membrane ruffles of the entry structure, which engulf entering bacteria, and also in the periphery of the phagosome early after bacterial internalization. The proto-oncoprotein pp60c-src appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the level of cortactin tyrosine phosphorylation induced during bacterial entry. Immunolocalization studies in pp60c-src-overexpressing HeLa cells indicate that pp60c-src is recruited to the entry structure and to the periphery of the phagosome, where pp60c-src appears to accumulate in association with the membrane. Our results suggest that epithelial cell invasion by S.flexneri involves recruitment and kinase activation of pp60c-src. Signalling by the proto-oncoprotein pp60c-src may play a role in cytoskeletal changes that facilitate S.flexneri uptake into epithelial cells, since transient overexpression of pp60c-src in HeLa cells can provoke membrane ruffling and appears also to stimulate bacterial uptake of a non-invasive S.flexneri strain.     

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.     

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.     

Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation.

The transforming protein of polyoma virus, middle T antigen, associates with the protein tyrosine kinase pp60c-src, and analysis of mutants of middle T suggests that this complex plays an important role in transformation by polyoma. It has recently been reported that pp60c-src from polyoma virus-transformed cells has enhanced tyrosine kinase activity in vitro. The data presented here confirm these findings and show that the enhanced kinase activity of pp60c-src is due to an increase in the Vmax of the enzyme. Sucrose density gradient analysis demonstrates that only the form of pp60c-src which is bound to middle T antigen is activated. The difference in enzyme activity between pp60c-src from normal and middle T-transformed cells is more marked when the enzyme is prepared from lysates containing the phosphotyrosine protein phosphatase inhibitor, sodium orthovanadate. pp60c-src from middle T transformed cells is unaffected, but pp60c-src from normal cells has reduced kinase activity if dephosphorylation is prevented. The kinase activity of pp60c-src thus appears to be regulated by its degree of phosphorylation at tyrosine, and data are presented which support this hypothesis. pp60c-src is the first example of a protein tyrosine kinase whose activity is inhibited by phosphorylation at tyrosine. Middle T antigen may increase the kinase activity of pp60c-src by preventing phosphorylation at this regulatory site.     

A protein tyrosine kinase involved in regulation of pp60c-src function

We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.     

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

The tyrosine protein kinase activities of pp60c-src and pp60v-src were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp60c-src was about 10-fold lower than that of pp60v-src for exogenous substrate phosphorylation but was only 1.1- to 2-fold lower than that of pp60v-src for autophosphorylation. Six glycolytic enzymes, including three not previously identified as substrates for pp60src phosphorylation, were phosphorylated by both pp60c-src and pp60v-src. Levels of pp60c-src fourfold higher than the amount of pp60v-src in src-plasmid-transformed cells did not detectably alter the level of phosphotyrosine in cellular proteins, but increasing the expression of pp60c-src another twofold (which induces cells to form foci in monolayer culture (P.J. Johnson, P.M. Coussens, A.V. Danko, and D. Shalloway, Mol. Cell. Biol. 5:1073-1083, 1985) resulted in a threefold increase in the level of cellular protein phosphotyrosine. Immunoprecipitation and analysis of the alkali-stable phosphoproteins by two-dimensional electrophoresis showed that, in contrast to pp60v-src-transformed cells, pp36 and enolase are only weakly phosphorylated in these high-level pp60c-src overexpresser cells. Even allowing for the in vitro differences in specific activities of phosphorylation, these results suggest that the pp60c-src tyrosine protein phosphorylating activity may be restricted relative to that of pp60v-src by additional in vivo mechanisms.     

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.     

Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts.

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.