Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold.
Initiation of cell migration requires morphological polarization with formation of a dominant leading pseudopodium and rear compartment. A molecular understanding of this process has been limited, due to the inability to biochemically separate the leading pseudopodium from the rear of the cell. Here we examine the spatio-temporal localization and activation of cytoskeletal-associated signals in purified pseudopodia directed to undergo growth or retraction. Pseudopodia growth requires assembly of a p130Crk-associated substrate (CAS)/c-CrkII (Crk) scaffold, which facilitates translocation and activation of Rac1. Interestingly, Rac1 activation then serves as a positive-feedback loop to maintain CAS/Crk coupling and pseudopodia extension. Conversely, disassembly of this molecular scaffold is critical for export and down regulation of Rac1 activity and induction of pseudopodia retraction. Surprisingly, the uncoupling of Crk from CAS during pseudopodium retraction is independent of changes in focal adhesion kinase activity and CAS tyrosine phosphorylation. These findings establish CAS/Crk as an essential scaffold for Rac1-mediated pseudopodia growth and retraction, and illustrate spatio-temporal segregation of cytoskeletal signals during cell polarization.
Cho
Samuel Y
SY
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Klemke
Richard L
RL
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D016764:Cell Polarity; D002630:Chemotactic Factors / Q000494:pharmacology; D002633:Chemotaxis / Q000502:physiology; D002522:Chlorocebus aethiops; D050719:Crk-Associated Substrate Protein; D004789:Enzyme Activation; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D050717:Retinoblastoma-Like Protein p130; D044767:Ubiquitin-Protein Ligases; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
C493842:Bcar1 protein, mouse; D002630:Chemotactic Factors; D050719:Crk-Associated Substrate Protein; D008246:Lysophospholipids; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D050717:Retinoblastoma-Like Protein p130; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; C067419:CBL protein, human; C493849:Cbl protein, mouse