Abstract
CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.
Highlights
Mammalian Crk-associated substrate (CAS), a major substrate of Src kinase, plays an important role in oncogenic transformation mediated by the v-crk and v-src oncogenes
CAS consists of an N-terminal Src Homology 3 (SH3) domain, a large central substrate domain (SD) formed by 15 repeats of the YxxP motif followed by a serine-rich domain, and a C-terminal part composed of binding sites for the SH2 and SH3 domains of Src (YDYVHL and RPLPSPP, respectively) and a CAS-family C-terminal homology domain
The interaction specificity was confirmed by phage Enzyme-linked immunosorbent assay (ELISA), and qualitative determination of relative binding affinities was performed for seven clones (Fig. 2B,C)
Summary
Mammalian Crk-associated substrate (CAS), a major substrate of Src kinase, plays an important role in oncogenic transformation mediated by the v-crk and v-src oncogenes (reviewed by ref. 1). The phosphorylation of tyrosines in SD by Src family kinases enables CAS interactions with the Crk and Nck adapters[6, 7] The extent of this phosphorylation is regulated by the CAS SH3 domain, which mediates the interaction of CAS with polyproline motifs of various kinases (FAK, PYK2/RAFTK, FRNK), phosphatases (PTP1B, PTP-PEST), and other proteins (C3G, CMS, CIZ and Vinculin)[8,9,10,11,12,13,14,15]. While the second binding pocket in all these SH3 domains is mostly hydrophobic, the CAS SH3 domain includes a negatively charged Glu[17] residue The presence of this unusual negatively charged cleft could contribute to the CAS SH3 domain specificity, perhaps recognizing unusual polyproline rich sequences. A systematic screen for CAS SH3 domain ligand preferences has not yet been performed, and despite the known structure of the CAS SH3 domain, the structural determinants of its regulation are not well understood
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