Phosphorylation of polyoma T antigens.

The T antigens of polyoma virus have been examined for phosphorylation in vivo and associated protein kinase activities in vitro. The 100K "large" T antigen is the major phosphoprotein among the T antigen species in vivo as determined by labeling virus-infected cells with 32P-orthophosphate. Hr-t mutants show normal phosphorylation of their 100K T antigens. The wild-type 56K plasma membrane-associated "middle" T antigen is also phosphorylated in the cell, but to a lesser extent than the 100K; this low level phosphorylation is also observed in the presumably altered 56K protein induced by hr-t mutant NG59 and in the 50K truncated "middle" T of hr-t mutant SD15. Addition of dibutyryl cyclic AMP to the medium does not affect labeling of either large or middle T antigens in wild-type- or mutant-infected cells. Thus no differences are observed in T antigen phosphorylation in vivo between wild-type virus and hr-t mutants. Hr-t mutants are defective in a protein kinase activity assayed in vitro by adding gamma-32P-ATP to T antigen immunoprecipitates. In the case of wild-type virus, the 56K protein is the major phosphate acceptor in the in vitro kinase reaction, with a somewhat lower level of phosphorylation observed in the 100K band. Hr-t mutants NG59 and SD15 show no labeling of the altered 56K or 50K, respectively, but do show detectable levels of 32P in the 100K bands. A wild-type virus carrying a small deletion affecting the 100K and 56k bands shows a normal level of kinase activity associated with the truncated T antigens. Ts-a mutants appear to be normal with respect to the middle T antigen-associated kinase. Photoaffinity labeling of infected cell extracts with 8-azido cyclic AMP shows that the two major classes of regulatory subunits of cyclic AMP-dependent protein kinases are present in the immunoprecipitates. Phosphorylation of histone H1 occurs when this substrate is added to immunoprecipitates of either mock-infected or virus-infected cells, again demonstrating the presence of cellular kinases. Further experiments will be required to determine whether the middle T antigen of polyoma virus is itself a protein kinase or simply a substrate for one or more cellular kinases.     

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.     

12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function.

The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.     

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.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells.

fyn is a member of the growing family of protein tyrosine kinase genes whose sequences are highly related to that of c-src. We have generated antibodies to peptides corresponding to two different amino-terminal sequences encoded by this gene. Antisera to both peptides recognized a 59 kd protein from human and mouse fibroblasts. p59fyn was phosphorylated in vivo on serine and tyrosine residues and was also myristylated. Furthermore, immune precipitates of p59fyn had tyrosine kinase activity in vitro, as measured by autophosphorylation and by phosphorylation of substrates such as enolase. This kinase activity was shown to be negatively regulated by tyrosine phosphorylation. We have also established that, like pp60c-src and p62c-yes, p59fyn was complexed with middle T antigen, the transforming protein of polyoma virus. However, the tyrosine kinase activity of p59fyn was not elevated in middle T transformed cells. Possible explanations for this are discussed.     

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.     

Phosphoinositide kinase activity and transformation

We have used the DNA tumor virus polyoma as a model system to examine whether the phosphatidylinositol (PI) turnover pathway is a critical target for transforming gene products. Polyoma-infected cells show elevated levels of polyphosphoinositides and polyphosphoinositols, and a PI kinase activity is associated with middle T antigen, a transforming gene product of polyoma virus. In anti-T immunoprecipitates from polyoma-infected or -transformed cells, comparisons of wild-type and polyoma mutants defective for transformation show a strong correlation between middle T-associated PI kinase activity and transforming ability. Middle T has previously been found to associate at the plasma membrane with pp60 c-src and to activate it as a tyrosine kinase. c-src itself does not appear to phosphorylate PI; however, the middle T/pp60 c-src tyrosine kinase activity may be important for activation of PI kinase. Ammonium orthovanadate, a tyrosine phosphatase inhibitor, elevates the middle T/pp60 c-src-associated PI kinase activity. We propose that middle T/pp60 c-src activates a PI kinase and modulates PI turnover in vivo by tyrosine phosphorylation.     

Sites of in vivo phosphorylation of the T cell tyrosine protein kinase in LSTRA cells and their alteration by tumor-promoting phorbol esters

The LSTRA cell line contains an elevated level of a tyrosine protein kinase of apparent molecular weight of 56,000 (pp56Tcell). Analysis of the tryptic fragments of this protein labeled in vivo with 32P shows that it contains four sites of tyrosine phosphorylation and one site of serine phosphorylation. Two of the sites of in vivo tyrosine phosphorylation are also labeled in vitro when membranes are incubated with [gamma-32P]ATP. One of the sites that is labeled in vivo and in vitro (site 1) is identical in sequence with the major site of tyrosine phosphorylation in the transforming protein of the Rous sarcoma virus. Analysis of the sites of in vivo phosphorylation in pp56Tcell from LSTRA cells treated with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) reveals that this agent induces at least four new sites of serine phosphorylation. Treatment with PMA also causes a selective reduction in the level of tyrosine phosphorylation in site 1. Thus PMA causes new sites of serine phosphorylation in pp56Tcell and reduces the amount of phosphate in one of the sites of tyrosine phosphorylation.     

An oligomeric form of simian virus 40 large T-antigen is immunologically related to the cellular tumor antigen p53.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.     

Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.     

The hr-t gene of polyoma virus

Malignant transformation of cells by polyoma virus results from the continual expression of a viral gene (hr-t) the normal function of which is to facilitate productive viral infection. The series of investigations described here on the polyoma hr-t gene originated with attempts to understand polyoma virus-cell interactions along lines suggested by temperate bacteriophage. Nucleic acid hybridization experiments indicated clearly that viral DNA persists in transformed cells and continues to be expressed. Radiobiological and other experiments, however, suggested a function for the expressed gene(s) which was not expected of a prophage: the promotion, rather than repression, of lytic virus growth. The hr-t gene acts pleiotropically to alter the physiological state of the host in a manner which facilitates virus production and induces a transformed cellular phenotype. The cellular alterations are manifested transiently during productive infection or abortive transformation, but permanently when the viral genome is integrated in stably transformed cells. hr-t mutants are defective in their growth in mice and in most cultured mouse cell lines. They are also unable to induce tumors or any of the morphological, structural, or growth-related changes which accompany cells transformation by the wild-type virus.The 22 kDa and 56 kDa proteins encoded in the early region of the viral DNA constitute dual products of the hr-t gene. hr-t mutants are localized in a narrow segment of the early region that specifies an amino acid sequence shared by these two overlapping proteins. Current efforts to link structural (i.e., mutational) changes with functional changes in these proteins center around the 56 kDa middle T antigen and its associated protein kinase activity. Assayed in vitro, this activity leads to phosphorylation of the 56 kDa protein itself, predominantly at a specific tyrosine residue in the C-terminal portion of the molecule. The middle T protein is anchored in cellular membranes by a hydrophobic tail close to the C-terminus. Membrane association is essential for transformation, as well as for the kinase activity. The common region of the 22 kDa/56 kDa proteins where hr-t mutants map has local regions of homology with highly conserved sequences in the pituitary glycoprotein hormones. The integrity of this region is also essential for transformation and for kinase activity. In vivo, the 56 kDa protein is a substrate for cellular kinase(s) and undergoes multiple phosphorylations (serine and/or threonine) that may affect the tyrosine-specific activity. These kinase reactions, originating in cellular membrane but potentially affecting pathways into the cytoplasm and nucleus, currently provide the most plausible biochemical mechanism underlying the pleiotropic effects of the hr-t gene.     

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.     

Polyoma T (tumor) antigen species in abortively and stably transformed cells

Stable neoplastic transformation of cells by polyoma virus requires the participation of two viral genes, designated ts-a and hr-t. The effects of mutations in these two genes on the patterns of T-antigen synthesis during productive infection have been previously described: ts- a mutants are affected in the “large” (100K) nuclear T antigen, and hr-t mutants are affected in the “middle” (36K, 56K, 63K) and “small” (22K) T agtigens. The latter are associated predominantly with the plasma membrane (56K) and cytosol fractions, rrespectively. Here we examine the expression of the various forms of polyoma T antigen in nonproductive infection (abortive transformation) as well as in stably transformed cell lines of different species. The results on abortive transformation are essentially the same as those described above for productive infection. In stably transformed cells, the middle and small T antigens are seen to various extents. The large T antigen, however, is often absent or present below the level of detection. Clones lacking the large T antigen are found most often among mouse transformants, but are also seen among rat transformants. Retention of the 100K species in transformed cells therefore appears to be, at least in part, an inverse function of the level of permissivity of the host toward productive viral infection. These findings indicate that the induction of the transformed phenotype in both abortively and stably transformed cells generally does not require the large T antigen, but rather the products of the hr-t gene.     

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.     

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.     

Activated Src tyrosine kinase phosphorylates Tyr-457 of bovine GTPase-activating protein (GAP) in vitro and the corresponding residue of rat GAP in vivo.

GTPase-activating protein (GAP) is a key regulator of the cellular Ras protein, which is implicated in oncogenic signal transduction pathways downstream of the viral Src (v-Src) kinase. Previous studies demonstrated that v-Src induces tyrosine phosphorylation of GAP, suggesting that GAP may provide a biochemical link between v-Src and Ras signaling pathways. To determine the precise residues in GAP phosphorylated by Src kinases, we used a baculovirus/insect cell expression system for investigating in vitro phosphorylation of GAP. Phosphopeptide mapping analysis revealed that v-Src and normal cellular Src (c-Src) phosphorylate tyrosine residues in bovine GAP at one major site and one minor site in vitro. Significantly, the major site of GAP phosphorylation in vitro is also the major site of in vivo tyrosine phosphorylation of GAP in rat fibroblasts transformed by v-Src. Analyses of GAP deletion mutants and TrpE-GAP fusion proteins established that Tyr-457 of bovine GAP (and the corresponding residue of rat and human GAP) is the major site of tyrosine phosphorylation. Our results demonstrate that the v-Src kinase induces phosphorylation of the same tyrosine residue of GAP in vitro and in vivo, suggesting that GAP is a direct substrate of activated Src kinases in vivo. Because epidermal growth factor receptor phosphorylates the equivalent tyrosine residue in human GAP (Tyr-460), these findings are consistent with the hypothesis that specific phosphorylation of GAP at this site may have a physiologically important role in regulating mitogenic Ras signaling pathways.     

Inhibition of v-Mos kinase activity by protein kinase A.

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.     

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.     

Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.