TY - JOUR
T1 - Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk
AU - Sriram, G.
AU - Jankowski, W.
AU - Kasikara, C.
AU - Reichman, C.
AU - Saleh, T.
AU - Nguyen, K. Q.
AU - Li, J.
AU - Hornbeck, P.
AU - Machida, K.
AU - Liu, T.
AU - Li, H.
AU - Kalodimos, C. G.
AU - Birge, R. B.
N1 - Publisher Copyright:
© 2015 Macmillan Publishers Limited.
PY - 2015/8/6
Y1 - 2015/8/6
N2 - Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.
AB - Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.
UR - http://www.scopus.com/inward/record.url?scp=84938973223&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84938973223&partnerID=8YFLogxK
U2 - 10.1038/onc.2014.361
DO - 10.1038/onc.2014.361
M3 - Article
C2 - 25381819
AN - SCOPUS:84938973223
SN - 0950-9232
VL - 34
SP - 4260
EP - 4269
JO - Oncogene
JF - Oncogene
IS - 32
ER -