Direct Binding of C-terminal Region of p130 to SH2 and SH3 Domains of Src Kinase

Tetsuya Nakamoto,Ryuichi Sakai,Keiya Ozawa,Yoshio Yazaki,Hisamaru Hirai

doi:10.1074/jbc.271.15.8959

Tetsuya Nakamoto, Ryuichi Sakai + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.15.8959

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Abstract

p130Cas is a major tyrosine-phosphorylated protein that tightly binds v-Crk in v-crk-transformed cells and v-Src in v-src-transformed cells. The "substrate domain" of p130Cas contains 15 possible Src homology (SH) 2-binding motifs, most of which conform to the binding motif for the Crk SH2 domain. Another region near its C terminus contains possible binding motifs for the Src SH2 domain and proline-rich sequences that are candidates for SH3-binding sites. Using GST fusion proteins, we revealed that both SH2 and SH3 domains of Src bind p130Cas, whereas v-Crk binds p130Cas through its SH2 domain. We located the binding site of p130Cas for the Src SH3 domain at the sequence RPLPSPP in the region near its C terminus. Mutations within this sequence or at Tyr762 of p130Cas caused a significant reduction in the association of p130Cas with Src, and no association was detected when both of them were deleted. The kinase activity in v-Crk-transformed cells was also associated with p130Cas through this region. On the other hand, the deletion of the substrate domain abolished the binding with v-Crk. The association through the C-terminal region of p130Cas with Src kinase may facilitate effective hyperphosphorylation of tyrosine residues in the substrate domain of p130Cas, resulting in the binding of SH2-containing molecules to p130Cas.

Highlights

Using GST fusion proteins, we revealed that both Src homology 2 (SH2) and Src homology 3 (SH3) domains of Src bind p130Cas, whereas v-Crk binds p130Cas through its SH2 domain
Proteins called “adapter proteins” such as Crk [2], Nck [12], and Grb2/Ash/Sem-5 [13,14,15] have only SH2 and SH3 domains and no catalytic domain. v-Crk, a transforming oncoprotein encoded by avian sarcoma viruses, is a fusion protein of viral gag protein and the SH2 and SH3 domains derived from c-Crk, a cellular counterpart of v-Crk [2]. v-Crk has an oncogenic potential and induces tyrosine phosphorylation of several proteins when expressed in fibroblasts, the mechanism of phosphorylation is unknown
The primary structure of p130Cas reveals that it has a proline-rich region and several tyrosine residues near its C terminus [18], and that these motifs fairly well conform to the consensus binding sequences for the SH2 and SH3 domains of Src

Summary

Introduction

Using GST fusion proteins, we revealed that both SH2 and SH3 domains of Src bind p130Cas, whereas v-Crk binds p130Cas through its SH2 domain. (18) was used for construction of a series of deletion mutants of p130Cas (Fig. 2A), since it could encode a 120-kDa gene product, which corresponds to the B form of p130Cas. These mutants were transiently expressed in COS-1 cells, and the cell lysates were reacted with immobilized GST-SrcSH3 fusion proteins to determine the binding site of the Src SH3 domain to p130Cas (Fig. 3A).

Results

Conclusion