The genome of CT10 avian sarcoma virus encodes a 47-kDa fusion protein that consists of viral gag sequences fused to a cell-derived sequence containing SH2 and SH3 domains (v-crk). Genetic and biochemical evidence suggests that v-Crk can induce transformation of chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulation. In this report, we have developed an in vitro microtiter assay to study the binding of bacterially expressed glutathione S-transferase-fusion proteins of v-Crk and its cellular homolog, c-Crk, to the phosphorylated epidermal growth factor receptor (EGFR). Competitive binding data are presented that compare the abilities of heterologous glutathione S- transferase-fusion proteins containing GAPSH2[N], AblSH2, SrcSH2, and PLC- γSH2[N] sequences to inhibit Crk binding. Results indicate that both full- length Crk and GAPSH2[N] bind the phosphorylated EGFR with high affinity and can quantitatively compete the binding of each other by competitive enzyme- linked immunosorbent assay. Binding of full-length Crk or the isolated SH2 domains of GAP or Abl resulted in a significant protection of phosphorylated EGFR against dephosphorylation by cellular phosphatase activity, but did not appear to stimulate the intrinsic tyrosine kinase activity of the EGFR. To extend these findings to p130, the major phosphotyrosine-containing protein in CT10-transformed cells, we utilized a nitrocellulose filter binding assay. Results demonstrate high affinity binding of Crk toward denatured p130 and, as is the case for phosphorylated EGFR, Crk binding can partially protect p130 from phosphatase activity. However, no apparent competition of Crk binding was noted with heterologous SH2-containing proteins including GAPSH2[N], suggesting a possible specificity of Crk-p130 binding. These data are consistent with a direct role of SH2 in the modulation of cellular phosphotyrosine status in vivo.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology