The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

Immunological characterization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus.

Phosphotyrosine antibodies were used to identify tyrosine-phosphorylated proteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. A large number of tyrosine phosphoproteins were detected. A similar set of proteins was observed in RSV-transformed murine cells. An 85,000-dalton protein, however, was present in transformed avian cells but missing in transformed murine cells. Neither the 85,000-dalton protein nor any of the other tyrosine phosphoproteins appeared to be viral structural proteins. Use of RSV mutants encoding partially deleted src gene products enabled us to identify a 60,000-dalton cellular tyrosine phosphoprotein that comigrated with wild-type pp60v-src. With the exception of calpactin I, the major tyrosine phosphoproteins detected in immunoblots appeared to be different from several previously characterized substrates of pp60v-src with similar molecular masses (ezrin, vinculin, and the fibronectin receptor).     

A tumor promoter stimulates phosphorylation on tyrosine

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate is mitogenic for normal chicken embryo fibroblasts and also causes these cells to express transiently many properties of cells transformed by Rous sarcoma virus. Since some mitogenic hormones stimulate a tyrosine-specific protein kinase activity, and since the transforming protein of RSV is a tyrosine-specific protein kinase, we have examined whether TPA also stimulates protein phosphorylation on tyrosine. We report here that TPA treatment of normal cells resulted in a very rapid phosphorylation on tyrosine of a protein peak of Mr 40 to 43 kilodaltons. Thus, a similar biochemical activity (tyrosine phosphorylation) is associated with the action of polypeptide mitogenic hormones, Rous sarcoma virus and a tumor promoter. In addition, TPA treatment resulted in rapid changes in phosphorylation of proteins on serine and threonine.     

Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus

We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000- dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor.

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.     

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.     

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

Isolation and characteristics of antibodies against synthetic fragments of oncoproteins. II. Antiserum against the transforming protein pp60src of Rous sarcoma virus

To generate the antibodies to the transforming protein of Rous sarcoma virus (RSV) pp60src, rabbits were immunized with the peptide, corresponding to 415-421 sequence of pp60src. These antibodies immunoprecipitate pp60src in RSV-transformed chicken and mammalian cells, and also some proteins (45, 85 and 120 kDa), which could be autophosphorylated in vitro. It was shown that 415-421 sequence of pp60src is not recognized by the antibodies to pp60src from RSV-induced tumour bearing rabbits (TBR serum). In contrast to TBR serum, antibodies, generated against synthetic peptide, corresponding 415-421 sequence of pp60src couldn't be phosphorylated in vitro, when [gamma-32P]ATP is added to the immune complex. The antipeptide antibodies, bound to pp60src did not block phosphorylation of TBR immunoglobulins, added to this immune complex. Hence, 415-421 sequence of pp60src RSV containing the major tyrosine phosphorylation site does not take part in the kinase reaction in vitro.     

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.     

A mouse homolog to the avian sarcoma virus src protein is a member of a protein kinase cascade

Recent work has identified a cascade of membrane bound protein kinases in Ehrlich ascites tumor cells. These enzymes, designated PKL, PKS and PKM, are present in both Ehrlich tumor and mouse brain, but the cascade is active only in the tumor tissue. We have now purified a fourth protein kinase, PKF, that is also associated with this cascade. Protein kinase F prosphorylates PKL and is phosphorylated by PKS. The position of this kinase in the cascade is as follows, where the arrows denote phosphorylation: [Formula: see text] The phosphorylation by PKF, like phosphorylation by the other kinases, is at a tyrosine residue and causes the substrate kinase (PKL) to become active. The role of the tyrosine phosphorylation in activating these kinases is described in detail elsewhere. One result of activation of the cascade is the phosphorylation of the beta subunit of the Na+K+-ATPase, which causes inefficient Na+ pumping and is at last in part responsible for the high aerobic glycolysis of Ehrlich ascites tumor cells. By several criteria protein kinase F from Ehrlich cells is homologous to the src gene product (pp60src) from avian sarcoma viruses. Antiserum raised against PKF and sera from rabbits bearing rous sarcoma virus (RSV)-induced tumors quantitatively precipitate the same 60 kd phosphoprotein from cell lysates of three different RSV-transformed cell lines. Both proteins phosphorylate PKL and a 130 kd cytoskeletal protein (vinculin). The tryptic maps of these proteins are closely similar. Both proteins bind specifically to PKL covalently coupled to Sepharose. We used this latter observation to facilitate the purification of pp60 src from RSV-transformed cells.     

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.     

Ecto-protein kinase activities in normal and transformed cells

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.     

Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells.

Rous sarcoma virus encodes a tyrosine-specific protein kinase (p60src) which is necessary for cell transformation. To identify substrates for this kinase, we set out to detect phosphotyrosine-containing proteins in Rous sarcoma virus-transformed chicken embryo cells, making use of the known alkali stability of phosphotyrosine. 32P-labeled phosphoproteins were separated by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then incubated in alkali. Using this procedure with normal cells, we detected a total of about 190 alkali-resistant phosphoproteins. In Rous sarcoma virus-transformed cells, five phosphoproteins were found which were not detectable in normal cells. Two of these are probably structural proteins of the virus. The other three transformation-dependent phosphoproteins, and four other phosphoproteins which were elevated by transformation, all contained phosphotyrosine. Increased phosphorylation of these proteins did not occur with cells infected with a mutant Rous sarcoma virus, temperature sensitive for transformation, grown at the restrictive temperature. We conclude that these seven proteins are probably substrates of p60src, although they may be substrates for other tyrosine-specific protein kinases activated by p60src.     

Interaction between the Rous sarcoma virus transforming protein and two cellular phosphoproteins: analysis of the turnover and distribution of this complex.

The transforming protein of Rous sarcoma virus (RSV), pp60src, was previously shown to associate with two cellular proteins of Mr 90,000 and 50,000 in RSV-transformed chicken cells. In this report, we demonstrate that this interaction is specific for a discrete population of pp60src molecules. Newly synthesized pp60src was found to preferentially associate with pp90 and pp50 to form a short-lived complex. The half-life of this complex varied from 9 to 15 min in cells transformed by nondefective strains of RSV. This interaction between pp60src, pp50, and pp90 took place in a soluble fraction of the cell, and the complex-bound pp60src molecules were not phosphorylated on tyrosine. These results suggest that pp90 and pp50 may be involved in the processing of pp60src molecules before the association of pp60src with the plasma membrane. The kinetics of dissociation of this complex were shown to be altered in cells infected with viruses containing a temperature-sensitive defect in the src gene. When cells infected with these viruses were grown at the nonpermissive temperature, more than 90% of the pp60src molecules were associated with pp90 and pp50, and little or no dissociation was observed in a 3-h chase period. These results suggest that mutations in the src gene which affect the transforming activity of pp60src also affect the stability of the interaction of pp60src with pp90 and pp50.     

Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts

We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high- speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells.     

Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.     

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.