Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts.

Abstract

The genome of avian sarcoma virus CT1O encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa yrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosinephosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. Although many additional cellular proteins became hyperphosphorylated on tyrosine in the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound preferentially 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 CT1O-CEF, and the elevation in tyrosine phosphorylation resulted in increased binding to GST-CrkSH2. Moreover, binding of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitro protected paxillin from dephosphorylation by cellular protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state 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.

PMA-induced phenotypic changes in K562 cells: MAPK-dependent and -independent events

“…This is supported by the observation that the presence of Pyk2 in CrkL immunoprecipitates or in Crk-SH2 in vitro co-precipitation experiments mirrors the pattern of tyrosine phosphorylated paxillin (Figures 2 and 4). Tyrosine phosphorylated paxillin is known to be a binding partner for the SH2 domain of CrkL-related proteins, 43,59 and is also found in a complex with Pyk2. 46 PMA-induced megakaryocytic differentiation of K562 cells is dependent on the activity of the MEK/MAPK pathway.…”

“…It has previously been shown that members of the CrkL family bind to phosphorylated paxillin. 43 The PMA-mediated events that take place upon stimulation of K562 cells lead to the expression of megakaryocyte markers. 8 Therefore, it was of interest to determine whether the expression of Pyk2, a tyrosine kinase expressed in megakaryocytes, 44,45 was regulated by PMA.…”

Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility

“…The abnormalities in lung cancer may also re¯ect changes in the cytoskeleton and cytoskeleton associated proteins. Previously, it has been shown that transforming oncogenes such as v-src (Brown and Cooper, 1996), v-crk (Birge et al, 1993), BCR/ABL , and E6 (bovine and human papilloma virus) (Tong and Howley, 1997) interact with the cytoskeleton in oncogenesis. It is therefore possible that the various molecular abnormalities in lung cancer also contribute to transformation via the cytoskeleton.…”

“…Paxillin is tyrosine phosphorylated in response to oncogenes such as v-src (Schaller and Parsons, 1994), v-crk (Birge et al, 1993) and BCR/ABL (Salgia et al, 1995a). This focal adhesion protein is tyrosine phosphorylated in response to growth factors such as PDGF/EGF (Rankin et al, 1996), IL-3 and steel factor (Salgia et al, 1995b), and neuropeptides such as bombesin (Charlesworth et al, 1996).…”

Commentary: The carboxyl‐terminal Crk SH3 domain: Regulatory strategies and new perspectives

“…Over the past two decades, enormous progress in our understanding of signal transduction has emerged from studies on Crk. The canonical signaling pathway, defined by recognition of specific phosphotyrosine motifs by the SH2 domain (in the context of p-Tyr.X.X.Pro) [4] and Polyproline Type II motifs by the N-terminal SH3 domain {in the context of Pro. X. X.…”