Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.     

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.     

Differential effects of polyamines on the phosphorylation of chromatin-associated proteins

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.     

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.     

Plasma membrane phosphoproteins in normal and Rous sarcoma virus transformed chick embryo fibroblasts: characterization by in vitro phosphorylation.

Plasma membranes isolated from normal and RSV transformed chick embryo fibroblasts were phosphorylated in vitro using endogenous protein kinase and ATP (gamma32P) and the labeled phosphoproteins were analyzed by SDS-PAGE. A number of protein phosphorylation changes were observed following transformation, however in most cases they were relatively small quantitative differences. The four major changes were in proteins of 47,000, 58,000, 75,000 and 135,000 daltons. Decreased phosphorylation of the 47,000 dalton polypeptide was found in transformed cell membranes but this alteration was shown to be due to differences in cell growth rather than transformation. Increase phosphorylation of the 75,000 dalton protein was at least partially related to virus infection. However, increased phosphorylation of the 58,000 and 135,000 dalton polypeptides were entirely transformation specific.     

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Inhibition by quercetin of the release of density dependent-inhibition of cell growth in RSV-transformed chicken cells

The expression of src gene in dense cultures of chick embryo fibroblasts (CEF) infected by a thermosensitive mutant (NY68) of RSV released density-dependent inhibition of growth and induced in these cells a large increase in DNA, RNA and protein synthesis. This stimulation of cellular metabolism was abolished in the presence of quercetin. Furthermore, quercetin added to the culture medium also inhibited the stimulation of pp60src kinase due to the expression of transformation.     

In vitro ADP-ribosylation of chromosomal proteins of the brain of developing rats

In vitro ADP-ribosylation of chromosomal protein and its modulation by spermine, 3-aminobenzamide (3-AB) and benzamide were studied by incubating the nuclei of cerebral hemisphere of 3-, 14- and 30-day old rats with ³²P-NAD⁺. Histones get ADP-ribosylated more than the non-histone chromosomal (NHC) protein. H1 is the major target for ADP-ribosylation. Among the nucleosomal histones, H2B is ADP-ribosylated most. The other core histones also get ADP-ribosylated to a lesser extent. ADP-ribosylation of both histones and NHC proteins decreases during development. Spermine stimulates, whereas 3-AB and benzamide inhibit, ³²P-ADP-ribose incorporation into histones and NHC proteins. These effects decrease with development. Mild digestion of chromatin by micrococcal nuclease (MNase), EcoRI, and AluI prior to ADP-ribosylation stimulates incorporation of ³²P-ADP-ribose. The degree of stimulation decreases as development proceeds. Such alterations indicate progressive condensation of chromatin with development.     

Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity.

Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.     

Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells.

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.     

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.     

Diverse mitogenic agents induce rapid phosphorylation of a common set of cellular proteins at tyrosine in quiescent mammalian cells

Protein phosphorylation of quiescent human skin fibroblasts was analyzed following stimulation by epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, serum, or 12-O-tetradecanoyl-phorbol-13-acetate. In mitogen-treated cells, a markedly increased phosphorylation of two Mr = 43,000 proteins and two Mr = 41,000 proteins was always detected by two-dimensional gel electrophoresis. More acidic forms were the dominant species and contained phosphotyrosine, phosphoserine, and phosphothreonine, while the basic forms contained phosphotyrosine and phosphoserine. The two Mr = 41,000 proteins were structurally related to each other. All mitogens seemed to stimulate the phosphorylation of each protein with the same site specificity. Induction of the same set of phosphoproteins was observed in mitogen-stimulated rat and mouse fibroblasts as well. These stimulated phosphorylations occurred rapidly, were maximal 5 min after exposure of cells to mitogens, and diminished gradually after 30 min. Mitogen-induced phosphorylation of these proteins was correlated to the extent of mitogen-stimulated DNA synthesis. In addition, such increased protein phosphorylation was not observed in exponentially growing cells, nor in Rous sarcoma virus-transformed rat cells. Thus, phosphorylation of the Mr = 43,000 and 41,000 proteins, which represents a common and specific response of cells to mitogens, could constitute an early event involved in the control of cellular G0----G1 transition.     

Interleukin 1 and tumour necrosis factor increase phosphorylation of the small heat shock protein. Effects in fibroblasts, Hep G2 and U937 cells

Interleukin 1 alpha and tumour necrosis factor-alpha stimulated phosphorylation of three 27 kDa phosphoproteins in MRC-5 fibroblasts which was sustained for up to 2 h after adding the cytokines. All three phosphoproteins were immunoprecipitated by a specific antiserum to the small mammalian heat shock protein, hsp 27. The three phosphoproteins from stimulated or control cells contained phosphoserine but not phosphothreonine or phosphotyrosine. Similar increases in phosphorylation of immunoprecipitable 27 kDa proteins were seen in U937 cells stimulated by TNF alpha and Hep G2 cells stimulated by IL1 alpha.     

A 41-kilodalton protein is a potential substrate for the p210bcr-abl protein-tyrosine kinase in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.     

Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies.

The Rous sarcoma virus (RSV) oncogene product pp60src is known to trigger the acquisition of the transformed phenotype by phosphorylating host cell target molecule(s) at tyrosine residues. To identify phosphotyrosine-containing proteins, rabbit antibodies were raised against the synthetic hapten p-azobenzene-phosphonate (ABP) that specifically cross-reacts with phosphorylated tyrosine. By immuno-decoration of proteins extracted from RSV-transformed mouse fibroblasts and transferred to nitrocellulose sheets, phosphoproteins of 130, 70 and 60 kd were identified. These molecules were found to be associated with the cellular fraction insoluble in non-ionic detergent. Moreover, ABP antibodies precipitated detergent-insoluble proteins of 130, 70 and 60 kd, plus two additional components of 85 and 65 kd, that had been phosphorylated in vitro by [gamma-32P]ATP under conditions allowing the kinase reaction catalyzed by pp60src. Phosphoproteins of closely related mol. wts. were immunoprecipitated from RSV-transformed avian fibroblasts. The radioactivity co-migrated with authentic phosphotyrosine in two-dimensional chromatography. The 60-kd protein comigrated with pp60src, while the identity between the 130-kd protein and vinculin was disproved by the lack of cross-reaction with appropriate antisera. In transformed mouse and duck fibroblasts ABP antibodies, employed in indirect immunofluorescence microscopy, stained diffusely the cytoplasm and intensely decorated restricted areas of the ventral cell plasma membrane. These data show that antibodies reacting with phosphotyrosine may be usefully employed in the identification and in the intracellular localization of molecules that are potential targets of the pp60src protein kinase.     

Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation

To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.     

Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol.

$\text{In}\text{vitro}$ phosphorylation and acetylation of nonhistone chromosomal (NHC) proteins and their modulation by Ca⁺⁺ and estradiol were studied by incubating slices of cerebral cortex of 2-, 15- and 84-week female rats with ³²Pi and ¹⁴C-Na-acetate. Phosphorylation pattern of NHC proteins is unique for each age. Ca⁺⁺ and estradiol stimulate phosphorylation of different NHC proteins which is also age-specific. Acetylation of NHC proteins decreases precipitously with age. No unique NHC protein is acetylated preferentially at any age, nor does Ca⁺⁺ stimulate acetylation. Estradiol, however, stimulates acetylation of a few NHC proteins. It is suggested that phosphorylation of NHC proteins and its modulation by effectors may be more important for gene expression than their acetylation.     

A protein kinase system from platelet rich plasma

Treatment of platelet rich plasma (PRP) at pH 5 results in the precipitation of a protein kinase system. The protein kinase is associated with the platelet fraction and is capable of phosphorylation of several plasma proteins. Analysis of the ³²P-labeled phosphoproteins by two dimensional gel electrophoresis showed the existence of three major phosphoproteins: 72K and 80K proteins with identical isoelectric points (pI) of 6.0 and another 72K protein with a pI of 6.8–7.0. This latter 72K phosphoprotein has recently been identified as the α-chain of fibrinogen. The identity of the other 2 proteins remains to be shown. The activity of the protein kinase is markedly enhanced by Mn²⁺, it phosphorylates calf thymus histone as an exogenous substrate and is independent of cAMP or cGMP. This protein kinase activity is inhibited competitively by ADP.     

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.     

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.