Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk

Oncogene. 2015 Aug 6;34(32):4260-9. doi: 10.1038/onc.2014.361. Epub 2014 Nov 10.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Cell Line, Tumor
  • Chromatography, Liquid
  • HEK293 Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • NIH 3T3 Cells
  • Oncogene Proteins v-abl / metabolism
  • Phosphorylation
  • Protein Binding
  • Proto-Oncogene Proteins c-crk / genetics
  • Proto-Oncogene Proteins c-crk / metabolism*
  • Sequence Homology, Amino Acid
  • Signal Transduction*
  • Tandem Mass Spectrometry
  • Tyrosine / genetics
  • Tyrosine / metabolism*
  • src Homology Domains*

Substances

  • CRK protein, human
  • Oncogene Proteins v-abl
  • Proto-Oncogene Proteins c-crk
  • Tyrosine