Vinculin: A cytoskeletal target of the transforming protein of rous sarcoma virus

Sefton, Bartholomew M.; Hunter, Tony; Ball, Eric H.; Singer, S. J.

doi:10.1016/0092-8674(81)90512-2

Cited by 598 publications

(313 citation statements)

References 59 publications

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“…Unfortunately, it is often observed that in vivo substrate and site specificities of protein kinases are not accurately reflected in ICK reactions. For instance, this phenomenon was described before for immune complexes containing pp6Osrc (3,26) and the middle T-antigen of polyomavirus (6,28). Interestingly, and in contrast to the related polyomavirus system, Tyr-specific phosphorylation appeared to be totally insignificant in the SV40 system both in vivo (7,33) and in vitro (15; this study).…”

Section: Resultsmentioning

confidence: 74%

Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Roy¹,

Fransen²,

Fiers³

1984

Mol. Cell. Biol.

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Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [-y-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

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Section: Resultsmentioning

confidence: 74%

Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Roy¹,

Fransen²,

Fiers³

1984

Mol. Cell. Biol.

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“…The majority of the transforming v-Src is located in cytoskeleton and phosphorylates the components of the cytoskeleton (Hamaguchi et al, 1987;Sefton et al, 1981;Pasquale et al, 1986;Gould et al, 1986;Wu et al, 1991;Turner et al, 1990). Normally, c-Src is associated with inner plasma membrane or with endosomal membrane, not cytoskeleton.…”

Section: Discussionmentioning

confidence: 99%

Tumorigenesis mediated by MET mutant M1268T is inhibited by dominant-negative Src

et al. 2000

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We recently described germline and somatic mutations in the MET gene associated with papillary renal carcinoma type 1. MET mutation M1268T was located in a codon highly conserved among receptor tyrosine kinases, and homologous to the codon mutated in multiple endocrine neoplasia type 2B, and many cases of sporadic medullary carcinoma of the thyroid gland (Ret M918T). Ret M918T and MET M1268T have previously been shown to be highly active in mouse NIH3T3 transformation assays, and to change the substrate speci®city of the kinase. We studied the mechanism of transformation mediated by MET M1268T by analysing a clone, F4, derived from NIH3T3 cells transformed by MET M1268T. In contrast to NIH3T3 cells, F4 cells grew in suspension in tissue culture, and rapidly formed tumors in nude mice. We found that c-Src was constitutively bound to MET proteins in F4 cells, and that Src kinase activity was elevated. Transfection of dominant negative Src constructs into F4 cells eliminated the ability of F4 cells to grow in suspension culture and retarded the growth of F4 cells in vivo. The ability of transfected dominant negative Src constructs to inhibit the growth of F4 cells correlated with the inhibition of phosphorylation of paxillin and focal adhesion kinase. Transfection of dominant negative Src constructs into F4 cells had no e ect on Grb2 binding or PLC g phosphorylation. The results suggest that c-Src participates in the tumorigenic phenotype induced in NIH3T3 cells by MET M1268T by signaling through focal adhesion kinase and paxillin.

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“…Possibly, phosphorylation and PIP2 binding are different steps in a signal transduction cascade or exert a cooperative effect on the vinculin conformation. Although in vivo the overall phosphorylation of vinculin is low [16], it could well be a critical step in the construction and remodelling of contact sites where only a small percentage of the cellular vinculin is involved at any given time point.…”

Section: Discussionmentioning

confidence: 99%

Intramolecular interactions regulate serine/threonine phosphorylation of vinculin

1996

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Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vincnlin is primarily phosphorylated within the tail domain and suggest a function of vincnlin phosphorylation in the regulation of the vinculin conformation.

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Vinculin: A cytoskeletal target of the transforming protein of rous sarcoma virus

Cited by 598 publications

References 59 publications

Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Tumorigenesis mediated by MET mutant M1268T is inhibited by dominant-negative Src

Intramolecular interactions regulate serine/threonine phosphorylation of vinculin

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