Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk.
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.
Department of Biochemistry and Molecular Biology, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
G
G
Sriram
Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA.
W
W
Jankowski
Department of Biochemistry and Molecular Biology, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
C
C
Kasikara
Department of Biochemistry and Molecular Biology, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
C
C
Reichman
Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA.
T
T
Saleh
Department of Biochemistry and Molecular Biology, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
K-Q
KQ
Nguyen
Cell Signaling Technology, Danvers, MA, USA.
J
J
Li
Cell Signaling Technology, Danvers, MA, USA.
P
P
Hornbeck
Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA.
K
K
Machida
Center for Advanced Proteomic Research, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
T
T
Liu
Center for Advanced Proteomic Research, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
H
H
Li
Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA.
C G
CG
Kalodimos
Department of Biochemistry and Molecular Biology, Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
R B
RB
Birge
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016374:Oncogene Proteins v-abl / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D050737:Proto-Oncogene Proteins c-crk / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
C485285:CRK protein, human; D016374:Oncogene Proteins v-abl; D050737:Proto-Oncogene Proteins c-crk; D014443:Tyrosine