Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk

Area of Science:

• Cellular signaling
• Protein phosphorylation
• Molecular biology

Background:

• Crk protein, a key regulator of signaling complex assembly, is controlled by phosphorylation at Y221.
• This Y221 phosphorylation creates an intramolecular auto-clamp, inhibiting SH2-N-terminal SH3 domain (SH3N) signaling.

Purpose of the Study:

• To investigate the role of Crk C-terminal SH3 domain (SH3C) phosphorylation.
• To elucidate the signaling mechanisms downstream of Crk phosphorylation.

Main Methods:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Generation of phospho-specific antibodies.
• High-throughput SH2 domain profiling.
• Bioinformatic approaches (artificial neural network, position-specific scoring matrix).

Main Results:

• Crk SH3C is phosphorylated at Y239 and/or Y251 by extracellular stimuli.
• Phosphorylation of SH3C creates a phosphoSH3C-SH3N unit, maintaining SH3N functionality.
• PhosphoSH3C binds to SH2 domains of proteins like Abl (via pY251) and C-terminal Src kinase (via pY239).
• PhosphoSH3C modulates Abl-mediated cell spreading and motility.

Conclusions:

• Crk phosphorylation at Y221 is not an off switch but redirects signaling.
• A novel phosphoSH3C-SH3N signaling axis is described, with SH3N as a common component.
• This mechanism highlights the versatility of Crk in signal transduction pathways.