Phosphorylation of Crk on tyrosine 251 in the RT loop of the SH3C domain promotes Abl kinase transactivation

Abstract

Here, we report the identification and characterization of a novel tyrosine phosphorylation site in the carboxy-terminal Src Homology 3 (SH3) (SH3C) domain of the Crk adaptor protein. Y251 is located in the highly conserved RT loop structure of the SH3C, a region of Crk involved in the allosteric regulation of the Abl kinase. Exploiting kinase assays to show that Y251 is phosphorylated by Abl in vitro, we generated affinity-purified antisera against phosphorylated Y251 in Crk and showed that Abl induces phosphorylation at Y251 in vivo, and that the kinetics of phosphorylation at Y251 and the negative regulatory Y221 site in vitro are similar. Y251 on endogenous Crk was robustly phosphorylated in chronic myelogenous leukemia cell lines and in A431 and MDA-MB-468 cells stimulated with epidermal growth factor. Using streptavidin–biotin pull downs and unbiased high-throughput Src Homology 2 (SH2) profiling approaches, we found that a pY251 phosphopeptide binds specifically to a subset of SH2 domains, including Abl and Arg SH2, and that binding of pY251 to Abl SH2 induces transactivation of Abl 1b. Finally, the Y251F Crk mutant significantly abrogates Abl transactivation in vitro and in vivo. These studies point to a yet unrealized positive regulatory role resulting from tyrosine phosphorylation of Crk, and identify a novel mechanism by which an adaptor protein activates a non-receptor tyrosine kinase by SH2 domain displacement.

SH3 domains: modules of protein–protein interactions

“…The SH3 domain of Src-family PTKs, which regulate many cellular functions, such as cell proliferation and differentiation, survival, migration and cytoskeletal modifications, is mainly involved in substrate recognition and downregulation of the kinase activity. Phosphorylation of the RT loop of SH3 domain in an adaptor protein can positively regulate kinase activity (Sriram et al 2011). In myosins, the SH3 domain regulates stability and motility.…”

“…SH2 kinase and SH2-SH3 linkers interact with the N-lobe and stabilize the active conformation of the kinase, whereas in the inactive molecule this interaction is absent due to rotation of the SH2 domain (Ogawa et al 2002). Phosphorylation of Y251 in the RT loop of the SH3C domain of Abl kinase results in transactivation of the Abl kinase (Sriram et al 2011). Phosphotyrosine-mediated signaling involves numerous targets that can be identified by such methods as global phosphoproteomic based on quantitative mass spectrometry (Guha et al 2008).…”

“…The SH2 and SH3 domains are involved in the downregulation or upregulation of the activity of c-Src family PTKs (Gmeiner and Horita 2001;Sriram et al 2011). The mechanism of regulation of several kinases, including Lck, Hck and c-Src, has been studied by crystallography and NMR (Gaul et al 2000), and the results suggest that along with phosphorylation of the activation loop, kinases may be regulated by binding or dissociation of their regulatory domains (Ghose et al 2001;Huse and Kuriyan 2002).…”

“…As one of six domains of Src-family PTKs, the SH3 domain carries out significant roles in substrate recognition, membrane localization and regulation of the kinase activity (Gmeiner and Horita 2001;Sriram et al 2011). SH3 domains bind proline-rich sequences, particularly those carrying the PxxP motif that have the lefthanded polyproline 2 (PPII) conformation (Mayer 2001;Musacchio 2002).…”

Commentary: The carboxyl‐terminal Crk SH3 domain: Regulatory strategies and new perspectives

“…The Crk SH3C, in contrast to conventional SH3 domains, has a tyrosine residue in the RT-loop structure. Recently, this residue, Y251, was shown to be phosphorylated by Abl and downstream of EGFR in addition to the negative regulatory tyrosine 221 [13]. Phosphorylation at Y251 enhances Abl activation by virtue of binding to the Abl SH2 domain and unclamping the auto-inhibited Abl kinase structure.…”