Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Hiroaki Tanaka,Ken-Ichi Akagi,Chitose Oneyama,Masakazu Tanaka,Yuichi Sasaki,Takashi Kanou,Young-Ho Lee,Daisuke Yokogawa,Marc-Werner Dobenecker,Atsushi Nakagawa,Masato Okada,Takahisa Ikegami

doi:10.1074/jbc.m112.439075

Abstract

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.

Highlights

Src homology 2 (SH2) domains are known to bind to phosphotyrosine followed by a few amino acids
Deletion mutants of Csk-binding protein (Cbp) used for the assay, Cbp3 [195–328], Cbp5 (289 –321), Cbp6 [302–321], and Cbp7 (312–321; Fig. 1A), were fused with GST and expressed in E. coli, and intact C-terminal Src kinase (Csk) was expressed in a baculovirus/insect Sf9 cell system
It is generally accepted that SH2 domains bind to ligands by recognizing a Tyr(P) along with the ϩ1 to ϩ3 amino acid residues that follow in the C-terminal direction

Summary

Background

Src homology 2 (SH2) domains are known to bind to phosphotyrosine followed by a few amino acids. Results: A novel interaction region was revealed by the solution structure of the C-terminal Src kinase SH2 domain in complex with the Csk-binding protein. We report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). For Csk to efficiently access SFKs, Csk interacts with the Csk-binding protein (Cbp/PAG), which is localized to membrane microdomains enriched in cholesterol, glycosphingolipids, and lipid rafts, and it is subsequently recruited to the reaction space [11, 12] This interaction is known to occur between the SH2 domain of Csk and the SFK-phosphorylated Tyr-314 of Cbp, but it remains unknown. We found that Csk recognizes the four canonical amino acids beginning with Tyr(P)-314 and a region on the N-terminal side of Tyr(P)-314 that contains Tyr-296

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION