Phosphorylation of a 36,000 Mr cellular protein in cells infected with partial transformation mutants of rous sarcoma virus.

We have isolated and characterized mutants of Rous sarcoma virus which induce some parameters of transformation but fail to fully induce other parameters. We believe these mutants code for a pp60src which phosphorylates some targets well but phosphorylates others poorly. Using these mutants, we examined the phosphorylation of a 36,000 Mr protein which is phosphorylated on a tyrosine in cells transformed by Rous sarcoma virus, in an attempt to correlate this phosphorylation with the expression of specific transformation parameters. We found that phosphorylation of the 36,000 Mr protein was neither necessary nor sufficient for loss of fibronectin or for loss of density-dependent inhibition of growth. Phosphorylation of the protein was not sufficient for morphological alterations, increased hexose transport, or loss of adhesiveness. For the parameters measured, the best correlation was with increased plasminogen activator. In addition, it is noteworthy that cells infected with the mutant CU2 displayed low levels of phosphorylation of the 36,000 Mr protein and also were deficient in anchorage-independent growth and tumorigenicity, raising the possibility that the phosphorylation of the 35,000 Mr protein may be required for malignant growth properties.     

Biochemical properties of p60v-src mutants that induce different cell transformation parameters.

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.     

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.     

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v- src, and implicates a pivotal role for polyamines in cell transformation.     

Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase

We have isolated mutants of Rous sarcoma virus from an unmutagenized stock of the Schmidt-Ruppin strain of Rous sarcoma virus. These mutants induce only a "partial" transformation, and the transformation properties induced show unusual properties or combinations. Cells infected with mutant CU2 have a unique "blebby" morphology, have lost surface fibronectin, form very small colonies in soft agar, and are nearly normal with respect to adhesiveness and hexose transport. Cells infected with mutant tsCU11 have a nearly normal morphology, but grow well in soft agar. Cells infected with mutant CU12 have a fusiform morphology, intermediate levels of hexose transport and fibronectin, and form very large colonies in soft agar. Because the appearance of the different parameters of transformation is dissociated in these mutant-infected cells, these data are interpreted as supporting a model in which the transforming protein pp60src interacts with more than one primary target in generating the transformed phenotype. All of the mutants display levels of pp60src kinase activity less than that of the wild type. In the case of mutant CU12, the lower kinase activity is in part a consequence of a lower steady-state amount of pp60src inside the cell.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.     

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.     

Molecular events leading to enhanced glucose transport in Rous sarcoma virus-transformed cells

Transformation by Rous sarcoma virus results in a dramatic increase in the rate at which the transformed cells transport glucose across the cell membrane. The increased transport rate is a consequence of an increased number of transporters in the transformed cells. Utilizing antibody raised against the purified human erythrocyte glucose transporter, we have identified the glucose transporter as a membrane glycoprotein with a monomer Mr of approximately 41,000. The increased rate of glucose transport is dependent on the activity of pp60src, the transforming protein of Rous sarcoma virus. This protein has been shown to be a protein kinase that phosphorylates on tyrosine residues. We have examined the tyrosine phosphorylation of a major cellular protein of Mr 36,000 in cells infected with a panel of partially transforming mutants of Rous sarcoma virus. One of these mutants (CU2) increases the rate of glucose transport only slightly and does not render the infected cells fully anchorage independent or tumorigenic (although other transformation parameters are fully induced). Cells infected with this mutant display a 36,000-dalton protein that is phosphorylated to a considerably lesser extent than cells infected with wild-type virus. Analyses of this sort may help to identify the cellular targets of pp60src whose phosphorylation is necessary for the increased glucose transport rate.     

Local mutagenesis of Rous sarcoma virus: The major sites of tyrosine and serine phosphorylation of p60src are dispensable for transformation

We have constructed mutants of Rous sarcoma virus expressing p60^src that are underphosphorylated on serine or tyrosine, by linker insertion or insertion/ deletion into cloned Rous sarcoma virus DNA, and recovery of mutant virus by transfection of chicken embryo fibroblasts. Cells infected with mutants whose p60^src lack the major site of either serine or tyrosine phosphorylation were morphologically transformed and formed colonies in soft agar. The tyrosine kinase activities of the mutant p60^src measured in vivo and in vitro were close to the wild type activity. Peptide mapping showed that phosphorylation on tyrosine and serine of p60^src is independent: the major phosphorylated tyrosine and the major phosphorylated serine can each be phosphorylated in the absence of phosphorylation of the other.     

Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-src

A mutant in src, the oncogene of Rous sarcoma virus, has been constructed in which the major phosphorylated tyrosine (Tyr-416, located in the carboxy-terminal half of the protein) has been replaced by phenylalanine. Mouse cells transformed with this mutant src form foci and grow in soft agar, indicative of a transformed state. Also, the mutant protein retains the wild-type ability to phosphorylate proteins on tyrosine. Partial proteolysis revealed that the carboxy-terminal half of the mutant protein was still phosphorylated, although apparently to a lesser extent. Analysis indicated that this residual phosphorylation was on tyrosine. We conclude that the major tyrosine phosphorylation in pp60v-src is not required for two of the protein's notable properties--protein kinase activity and transformation of cultured cells.     

Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies.

The protein substrates for the tyrosine protein kinases in cells transformed by avian sarcoma viruses were analyzed by gel electrophoresis in combination with immunoblotting or immunoprecipitation by antibodies against phosphotyrosine. We found that greater than 90% of phosphotyrosine-containing cellular proteins can be immunoprecipitated by these antibodies. The level of phosphotyrosine-containing cellular proteins detectable by this method markedly increased upon transformation with Rous sarcoma virus, and more than 20 distinct bands of such proteins were found in lysates of Rous sarcoma virus-transformed cells. Most of these phosphotyrosine-containing proteins had not been identified by other methods, and their presence appeared to correlate with morphological transformation in cells infected with various Rous sarcoma virus mutants and Y73, PRCII, and Fujinami sarcoma viruses. However, considerably different patterns were obtained with cells infected with nontransforming Rous sarcoma virus mutants that encode nonmyristylated src kinases, indicating that most substrates that correlate with transformation can only be recognized by p60v-src associated with the plasma membrane.     

A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.

We have constructed mutants by using linker insertion followed by deletion in the region of cloned Rous sarcoma virus DNA coding for the N-terminal 9 kilodaltons of the src protein. Previous work implicated this region in the membrane association of the protein. The mutations had little effect on src tyrosine kinase activity. Substitution of a tri- or tetrapeptide for amino acids 15 to 27, 15 to 49, or 15 to 81 had little effect on the in vitro transforming capacity of the virus. Like wild-type p60src, the src proteins of these mutants associated with plasma membranes and were labeled with [3H]myristic acid. In contrast, a mutant whose src protein had the dipeptide Asp-Leu substituted for amino acids 2 to 81 and a mutant with the tripeptide Asp-Leu-Gly substituted for amino acids 2 to 15 were transformation defective, and the mutant proteins did not associate with membranes and were not labeled with [3H]myristic acid. These results suggest that amino acids 2 to 15 serve as an attachment site for myristic acid and as a membrane anchor. Since deletions including this region prevent transformation, and since tyrosine kinase activity is not diminished by the deletions, these results imply that target recognition is impaired by mutations altering the very N terminus, perhaps through their effect on membrane association.     

Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus

Vinculin, a protein associated with the cytoplasmic face of the focal adhesion plaques which anchor actin-containing microfilaments to the plasma membrane and attach a cell to the substratum, contains 8-fold more phosphotyrosine in cells transformed by Rous sarcoma virus than in uninfected cells. Because the transforming protein of RSV, p60src, is a protein kinase that modifies cellular proteins through the phosphorylation of tyrosine and because phosphotyrosine is a very rare modified amino acid, this result is a very rare modified amino acid, this result suggests that vinculin is a primary substrate of p60src. Only trace amounts of phosphotyrosine were detected in myosin heavy chains, alpha-actinin, filamin, and the intermediate filament protein vimentin. The modification of vinculin by p60src may be responsible in part for the disruption of the microfilament organization and for the changes in cell shape and adhesiveness which accompany transformation by Rous sarcoma virus.     

The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity.

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.     

Relationship between changes in the calcium dependent regulatory protein and adenylate cyclase during viral transformation.

The levels of the calcium dependent regulatory protein in transformed chicken embryo fibroblasts are higher both in soluble fractions and membrane fractions compared to untransformed cells. The kinetics for changes in the calcium dependent regulatory protein, hexose transport, and adenylate cyclase were compared using a temperature sensitive mutant of Rous sarcoma virus. Decreases in adenylate cyclase activity and increased hexose transport accompanying transformation occurred with half-lives of approximately 7 to 8 hours. Increases in the calcium dependent regulatory protein occurred much slower with a half-life of seventeen hours. It is concluded that the increase in calcium dependent regulatory protein levels is a late event during viral transformation and that the decline in adenylate cyclase activity cannot be due to changes in the amount of calcium dependent regulatory protein.     

Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus

Recent studies from this laboratory have identified novel cytoskeletal proteins that are phosphorylated on tyrosine in vivo in Rous sarcoma virus-transformed chick fibroblasts (Glenney, J. R., Jr., and Zokas, L. (1989) J. Cell Biol. 108, 2401-2408). In the present report, the phosphorylation of these proteins was examined in cells expressing the nonmyristylated mutants of src that are not transformed. A good correlation was found between transformation and the tyrosine phosphorylation of a 22-kDa protein. Tyrosine phosphorylation of the 22-kDa protein was reduced more than 95% in cells expressing the nonmyristylated mutants of src. Size fractionation revealed that the 22-kDa phosphoprotein in transformed chick fibroblasts is found in a Mr 150,000 complex. Monoclonal antibodies were used to screen various chicken tissues where the 22-kDa protein was found at high levels in muscle and lung with low levels in epithelial cells and brain. The 22-kDa protein becomes an excellent candidate for a mediator of transformation by the tyrosine kinase class of oncogenes.     

Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60^src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60^src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.     

Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus

Fujinami sarcoma virus (FSV), a newly characterized avian sarcoma virus, produces a protein of 140,000 daltons (p140) in infected cells. p140 is the product of a fused gene consisting of a part of the gag gene of avian retrovirus and FSV-unique sequences which are not related to the src sequences of Rous sarcoma virus. In vivo, p140 was found to be phosphorylated at both serine and tyrosine residues. Immunoprecipitates of p140 with antiserum against gag gene-coded proteins had a cyclic nucleotide-independent protein kinase activity which phosphorylated p140 itself, rabbit IgG of the immune complex and alpha-casein, an externally added soluble protein substrate. The phosphorylation was specific to tyrosine of the substrate proteins. p140 was phosphorylated in vitro at the same two tyrosine residues that were phosphorylated in vivo. The phosphate transferred to tyrosine residues of p140 forms a stable bond: it does not turn over during the kinase reaction, and the 32P-phosphate of p140 labeled in vitro or in vivo is not transferred to alpha-casein. FSV-p140 differs from p60src, the transforming protein of Rous sarcoma virus, in its marked preference of Mn2+ to Mg2+ ions, and in its inability to use GTP instead of ATP as the donor of gamma-phosphate.     

Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells.

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.