C-terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac-GEF.
Genetic studies in Caenorhabditis 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.
Akakura
Shin
S
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA.
Kar
Bishnupriya
B
Singh
Sukhwinder
S
Cho
Leong
L
Tibrewal
Nitu
N
Sanokawa-Akakura
Reiko
R
Reichman
Charles
C
Ravichandran
Kodimangalam S
KS
Birge
Raymond B
RB
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D016296:Mutagenesis; D041681:NIH 3T3 Cells; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C099818:DOCK1 protein, human; C465214:ELMO1 protein, human; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C480633:SLC2A4RG protein, human; D014157:Transcription Factors; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein