IDENTIFICATION AND CHARACTERIZATION OF A HIGH-AFFINITY INTERACTION BETWEEN V-CRK AND TYROSINE-PHOSPHORYLATED PAXILLIN IN CT10-TRANSFORMED FIBROBLASTS

Citation
Rb. Birge et al., IDENTIFICATION AND CHARACTERIZATION OF A HIGH-AFFINITY INTERACTION BETWEEN V-CRK AND TYROSINE-PHOSPHORYLATED PAXILLIN IN CT10-TRANSFORMED FIBROBLASTS, Molecular and cellular biology, 13(8), 1993, pp. 4648-4656
Citations number
49
Categorie Soggetti
Biology
ISSN journal
02707306
Volume
13
Issue
8
Year of publication
1993
Pages
4648 - 4656
Database
ISI
SICI code
0270-7306(1993)13:8<4648:IACOAH>2.0.ZU;2-W
Abstract
The genome of avian sarcoma virus CT10 encodes a fusion protein in whi ch viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Trans formation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specif ic cellular proteins with molecular weights of 130,000, 110,000, and 7 0,000 (p130, p110, and p70, respectively), an event which has been cor related with cell transformation. In this study, we have identified th e 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10 -CEF) as paxillin, a cytoskeletal protein suggested to be important fo r organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunopreci pitation studies. Moreover, a bacterially expressed recombinant glutat hione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is s ufficient for binding. Mapping of the sequence specificity of a GST-Cr kSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipit ation studies, an acetylated A(p)YDAPA hexapeptide was able to quantit atively inhibit the binding of GST-CrkSH2 to paxillin and p130, sugges ting that it meets the minimal structural requirements necessary for t he interaction of CrkSH2 with physiological targets. To investigate th e mechanism by which v-Crk elevates the tyrosine phosphorylation of pa xillin in vivo, we have treated normal CEF and CT10-CEF with sodium va nadate to inhibit protein tyrosine phosphatase activity. Although many additional cellular proteins became hyperphosphorylated on tyrosine i n the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound p referentially to the paxillin and 130-kDa proteins, suggesting a high degree of specificity in the interaction of CrkSH2 with these proteins . Paxillin phosphorylation was highly sensitive to vanadate treatment in both normal CEF and CT10-CEF, and the elevation in tyrosine phospho rylation resulted in increased binding to GST-CrkSH2. Moreover, bindin g of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitr o protected paxillin from dephosphorylation by cellular protein tyrosi ne phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and tha t v-Crk binding may interrupt this balance to increase the steady-stat e level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.