Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling.
The adaptor protein CrkII plays a central role in signal transduction cascades downstream of a number of different stimuli. We and others have previously shown that CrkII mediates attachment-induced JNK activation, membrane ruffling and cell motility in a Rac-dependent manner. We report here that cell attachment leads to tyrosine phosphorylation of CrkII on Y221, and that CrkII-Y221F mutant demonstrates enhanced association with the Crk-binding partners C3G and paxillin. Despite this enhanced signaling complex formation, CrkII-Y221F fails to induce JNK and PAK activation, membrane ruffling and cell migration, suggesting that it is defective in activating Rac signaling. Wild-type CrkII has no effect on adhesion-induced GTP loading of Rac, but its expression results in enhanced membrane localization of Rac, which is known to be required for Rac signaling. In contrast, CrkII-Y221F is deficient in enhancing membrane localization of Rac. Mutations in Rac and CrkII-Y221F that force membrane targeting of these molecules restore Rac signaling in adherent cells. Together, these results indicate that the Y221 site in CrkII regulates Rac membrane translocation upon cell adhesion, which is necessary for activation of downstream Rac signaling pathways.
Cancer Research Center, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
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D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000648:ultrastructure; D002465:Cell Movement; D022081:Cell Surface Extensions / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005353:Fibronectins; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D050737:Proto-Oncogene Proteins c-crk; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry / Q000502:physiology; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D018909:src Homology Domains
C493872:Crk protein, rat; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D020735:Guanine Nucleotide-Releasing Factor 2; D008565:Membrane Proteins; D051419:Paxillin; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C496063:Pxn protein, rat; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins