Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor

Abstract

The Crk proto-oncogene product is an SH2 and SH3 domaincontaining adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in itro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in itro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in itro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in itro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which

c-Crk, a Substrate of the Insulin-like Growth Factor-1 Receptor Tyrosine Kinase, Functions as an Early Signal Mediator in the Adipocyte Differentiation Process

“…7C, panel II). Taken together, these results provide strong evidence that pp35 is the tyrosine-phosphorylated form of mouse c-Crk, a known substrate of the IGF-1 receptor tyrosine kinase (39,40).…”

Interplay of the Proto-oncogene Proteins CrkL and CrkII in Insulin-like Growth Factor-I Receptor-mediated Signal Transduction

“…Recently, we demonstrated in vitro that the SH2 domain of CrkII interacted with the phosphorylated tyrosines of the juxtamembrane region of IGF-I receptor and especially with tyrosine 950 (51). Based on these data we speculate that CrkII but not CrkL may compete with IRS-I but not with IRS-2 for binding of tyrosine 950 of the IGF-I receptor.…”

“…However, the significantly lower level of tyrosine phosphorylation of CrkII compare with CrkL after IGF-I stimulation of 293 cells, the inability of CrkL to inhibit this phosphorylation, the direct interaction of CrkII with the IGF-I receptor in vitro (51), and the possible competition of CrkII with IRS-I for binding to the receptor may indicate that the direct association of CrkII with the IGF-I receptor may contribute, at least in part, to IGF-I-dependent phosphorylation of CrkII in 293 cells. In fact, in order for GST-CrkII fusion proteins to be efficiently phosphorylated by purified IGF-I receptors in the in vitro kinase assay, the SH2 domain of CrkII must be present (51). Localization of Crk proteins-binding sites on IRS-4 are necessary to verify these hypotheses.…”

Crk family adaptors–signalling complex formation and biological roles

“…Subsequently, several reports accumulated evidence using cell culture models which support this hypothesis, initially based only on biochemical in vitro data. In response to EGF, insulin or NGF-treatment of certain cells, a phosphorylation of c-Crk II on Y221 which is followed by dissociation of complexes mediated by the SH3(1) domain of c-Crk II has been repeatedly found (Beitner-Johnson and LeRoith, 1995;Okada et al, 1997Okada et al, , 1998Koval et al, 1998a;Hashimoto et al, 1998;Nakashima et al, 1999;Escalante et al, 2000;Kain and Klemke, 2001). These events are probably cell type-speci®c and can be only analysed on a case by case basis.…”