Abstract
Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.
Highlights
Src protein-tyrosine kinases (PTK)2 are important enzymes in several signal transduction pathways initiated by a variety of cell surface receptors, such as receptor tyrosine kinases [1], cytokine receptors and antigen receptors [2], and G-protein-coupled receptors [3]
A Phosphopeptide Mimicking the Phosphorylated C-terminal Tail of Src Has Weak Affinity for the Src SH2 Domain—The C-terminal tail of Src has the sequence of Pro-Gln-Tyr527-GlnPro-Gly, which is significantly different from the optimal phosphopeptide ligand for the Src SH2 domain, pYEEI [15]
We characterized this interaction by measuring the binding of the phosphopeptide, Pro-Gln-Tyr(P)527-Gln-Pro-Gly, to the SH2 domain in a kinase-defective mutant of Src. kdSrc contains an inactivating point mutation (K295M) and lacks the myristoylation motif and the unique region at the N terminus
Summary
Src protein-tyrosine kinases (PTK)2 are important enzymes in several signal transduction pathways initiated by a variety of cell surface receptors, such as receptor tyrosine kinases [1], cytokine receptors and antigen receptors [2], and G-protein-coupled receptors [3]. Phosphorylation of kdSrc-R175A by Csk further reduced its affinity for Flu-GpYEEI, indicating that the mutant SH2 domain was still able to bind to Tyr(P)527. To further test this conclusion, we introduced the mutation to the SH2 domain of active Src, and we determined whether this mutant (Src-R175A) was still inactivated by Csk phosphorylation.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.