C‐terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac‐GEF

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Abstract
Genetic studies in Caenorhabiditis elegans identified an evolutionarily conserved CED‐2 (CrkII), CED‐5 (DOCK180), CED‐12 (ELMO), CED‐10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain‐independent Rac guanine nucleotide exchange factor (Rac‐GEF); but it is still unclear how CrkII functions in Rac‐GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C‐terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C‐terminal SH3 domain of CrkII (CrkII‐SH3‐C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac‐GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3‐N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N‐terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C‐terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac‐GEF.
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