The ability of Crk to function as an adaptor protein is negatively regulated and terminated by phosphorylation on Y221, which results in an intramolecular SH2-pTyr clamp, thereby resulting in the disassembly of Crk-mediated signaling complexes (Feller et al., 1994
; Rosen et al., 1995
; Kobashigawa et al., 2007
). Here, we show that in addition to Y221, another tyrosine residue, namely Y251, located within the highly conserved RT loop of the SH3C domain of Crk is also concomitantly phosphorylated by the Abl and Bcr-Abl tyrosine kinases. Our present data amend our current understanding of the role that tyrosine phosphorylation of Crk has in signal transduction, and clearly point to more elaborate and dynamic regulatory networks controlling the interaction between Crk and Abl.
Prompted by observations that Y221F Crk is phosphorylated by Abl using in vitro
kinase assays, we set out to identify additional tyrosine phosphorylation sites on Crk. As the PNAY motif in the RT loop of SH3C was essential for Crk-mediated Abl transactivation (Reichman et al., 2005
), we focused on Y251 which was a part of the PNAY sequence. In this study, we found that Y251 is phosphorylated by Abl, in Bcr-Abl-expressing CML cells and in A431 and MDA-MB-468 cells stimulated with EGF. In addition, we show that a 16-mer phosphopeptide flanking Y251 on Crk binds in trans
to Abl SH2, and in doing so, stimulates the kinase activity of Abl. Consistent with this interpretation, co-expression of the Y251F Crk mutant with Abl 1b partially suppressed Abl activation, and also purified GST-hCrk Y251F had a significantly attenuated ability to transactivate Abl compared with GST-hCrk. In addition, GST-hCrk failed to transactivate the Abl SH2 domain mutant R171L, suggesting that SH2 displacement by phospho (Y251) comprises one important part of the mechanism for Abl transactivation by hCrk.
Despite the fact that pY251 binds selectively to the Abl SH2 domain, it is noteworthy that the sequence around phospho (Y251) in hCrk ([pY251
DKT]) does not conform to the experimentally determined consensus peptide-binding motif for the Abl SH2 domain (pY[E/T/M][N/E/D][P/V/L]) (Birge et al., 1993
; Songyang et al., 1993
), In addition, Abl SH2 was not the strongest binding partner of pY251 in the SH2 domain screen (four-fold lower binding than the Arg SH2 in the assay), and isothermal titration calorimetry revealed a low-affinity interaction between pY251 and Abl SH2. However, as Crk binds to Abl via the SH3N domain (Feller et al., 1994
; Ren et al., 1994
), phospho (Y251) on hCrk and Abl SH2 would be expected to be present at high local concentrations in Abl–Crk complexes that may override the apparent low affinity and drive binding of the phospho (Y251) motif to Abl SH2. Interestingly and consistent with this notion, we observe a modest reduction in the amount Abl that co-immunoprecipitates with hCrk Y251F compared with WT hCrk when each is co-expressed with Abl in 293T cells (Supplementary Figure S4
), suggesting that the phospho (Y251)–Abl SH2 interaction, in addition to the Crk SH3N–Abl PXXP interaction, contributes to the stoichiometry of binding in Abl–Crk complexes.
Using in vitro
kinase assays to reconstitute Crk and Abl in vitro
, we observed that both Y221 and Y251 were phosphorylated at the earliest time point (5 s) examined. Therefore, it is not clear at the molecular level whether phosphorylation at Y251 (the transactivating phosphorylation) precedes phosphorylation at Y221 (the autoinhibition phosphorylation). Although it might be predicted that transactivation precedes inhibition, the present data suggest that both events occur quite rapidly and are likely in dynamic equilibrium, possibly in a manner to fine-tune Abl activation and its concomitant inhibition. Our data also imply that a pY221/pY251 dual phosphorylated species of Crk exist in cells. This predicts an interesting scenario in which Crk pY221/pY251 could dissociate from Abl by virtue of the negative regulation and pY251 binds in trans
to other SH2/PTB-containing proteins. Indeed, in SH2 profiling screens, we found evidence that pY251 binds in trans
to other SH2 domains selectively, which include ShcB, Brdg1 and Ship2. This interpretation would be consistent with previous reports that pY221 Crk retains biological activity (Abassi and Vuori, 2002
), possibly by virtue of its ability to engage in new protein complexes after dissociation from Abl. Clearly, the identification of in vivo
pY251 Crk containing complexes is an important future endeavor.
An equally important area will be the identification of additional tyrosine kinases, besides Abl, which can phosphorylate Y251. Using bioinformatic tools such as NetPhos (ExPaSy Proteomics Server, Swiss Institute of Bioinformatics, http://expasy.org
), no kinases were predicted to phosphorylate Y251, although in our study, we show that both EGFR and Abl can induce phosphorylation at Y251, at least when these kinases are overexpressed in cancer cells. Hence, our expectation is that Abl will not be the sole kinase capable of phosphorylating Y251 in vivo
, but rather we anticipate that multiple kinases may converge on this motif, hence integrating multiple upstream pathways with Abl. Recent evidence suggests that Abl is activated in aggressive breast cancer cell lines (includingMDA-MB-468) and possibly promotes cell invasion (Srinivasan and Plattner, 2006
; Srinivasan et al., 2008
). In light of our results that reveal phosphorylation of Crk at Y251 upon EGF stimulation of MDA-MB-468 cells and the fact that Abl is activated downstream of activated EGFR (Plattner et al., 1999
; Jones et al., 2006
), phospho (Y251) on Crk may be an important mediator of Abl activation downstream of EGFR. However, as activated EGFR also phosphorylates Crk at the negative regulatory tyrosine Y221 (Hashimoto et al., 1998
), the relative stoichiometry of phosphorylation at Y251 and Y221 might be critical as a high pY251/pY221 ratio on Crk typically induced by activated EGFR could favor Abl activation, and may resolve the issue of when Abl induces a tumor-suppressing signal (Noren et al., 2006
) versus a tumor-promoting signal (Srinivasan and Plattner, 2006
). In addition, it also remains to be determined whether Y251 is phosphorylated in human cancers as Crk has been shown to be overexpressed in several human cancers and knockdown experiments suggest that at least one of its roles is to promote cell migration (Nishihara et al., 2002
; Miller et al., 2003
; Rodrigues et al., 2005
; Linghu et al., 2006
; Wang et al., 2007
; Fathers et al., 2010
). Notably, in Crk (−/−) mouse embryonic fibroblasts (MEFs) stably overexpressing enhanced yellow fluorescent protein (EYFP), EYFP-hCrk or EYFP-hCrk Y251F, WT hCrk and the Y251F mutant equally enhanced cell spreading on fibronectin, and additionally, there was no significant difference in cell migration towards high serum in a transwell migration assay between WT hCrk- and Y251F-expressing cells (data not shown), suggesting that the Y251F mutant does not behave as a dominant-negative protein. Phosphorylation of hCrk at Y251 could be a gain-of-function modification downstream of specific stimuli such as EGF, by means of which non-canonical signaling pathways are engaged by Crk.
Our present observations may also have relevance to explain functional differences between Abl activation by Crk versus CrkL. It is intriguing that the PNAY251
motif in Crk SH3C is unique in all 266 known SH3 domains, and even diverges to a PCAY motif in CrkL. In addition to the PNAY in the RT loop of CrkSH3C, previous studies from Pawson’s laboratory showed that the hCrk SH2 domain contains an extended loop (called the DE loop) that contains a PRPP motif, which binds in trans
to the Abl SH3 domain (Anafi et al., 1996
). As such, double occupancy of the Abl SH2 domain with pY251 (in the RT loop of SH3C) and that of the Abl SH3 domain (by PRPP in the DE loop of the SH2 domain) may be required for full activation of Abl by hCrk. It is also noteworthy that such a proline-rich insert is lacking in CrkL, suggesting another important difference between Crk and CrkL (), and it will be interesting to test the combined effects of pY251 and PRPP peptides in the aforementioned kinase assays to ascertain cooperation between these motifs in Crk.
Model for the transactivation of Abl 1b by Crk. (a) Location of Y251 in Crk based on the NMR-derived structure (PDB ID: 2EYZ). (b) Multiple Crk/Abl interactions and the proposed mechanism for Crk-mediated Abl transactivation is shown.