Abstract
Induction of epithelial cell motility is a fundamental morphogenetic event that is recapitulated during carcinoma metastasis. Random motility of NBT-II carcinoma cells on collagen critically depends on paxillin phosphorylation at Tyr-31 and Tyr-118, the binding sites for the adapter protein CrkII. Two constitutive partners of CrkII are the exchange factors DOCK180 and C3G. CrkII bound to DOCK180 formed a signaling complex with phosphorylated paxillin that was necessary for cell migration as inferred from the inhibition caused by a DOCK180-interfering mutant. DOCK180, which acts predominantly on the Rho family GTPase Rac1, restored cell locomotion in cells expressing Phe-31/118 paxillin mutants deficient in Rac1 GTP-loading, suggesting that formation of paxillin-Crk-DOCK180 signaling complex controls collagen-dependent migration mainly through Rac1 activation. In migrating cells, CrkII constitutive association with C3G was not sufficient to stimulate its GDP/GTP exchange activity toward the Ras family GTPase Rap1. However, when constitutively active RapV12 was overexpressed, it negatively regulated cell motility. Activation of the C3G/Rap1 signaling pathway resulted in down-regulation of the paxillin-Crk-DOCK180 complex and reduction of Rac1-GTP, suggesting that Rap1 activation could suppress the Rac1 signaling pathway in epithelial cells.
Highlights
One efficient way to regulate complex cellular behaviors such as cell migration is to assemble functional networks composed of multidomain proteins [1]
The guanine nucleotide exchange factor (GEF) DOCK180 is among the proteins that has been previously identified to associate with the N-terminal SH3 domain of Crk [9]
CrkII-DOCK180 Signaling Complex Binds to Paxillin in NBT-II Cells—To identify signaling partners downstream of the paxillin-Crk complex, we carried out glutathione S-transferase (GST) pull-down assays using the N-terminal SH3 domain of Crk on protein extracts obtained from cells stimulated with collagen or PL, a control for integrin-independent cell adhesion
Summary
One efficient way to regulate complex cellular behaviors such as cell migration is to assemble functional networks composed of multidomain proteins [1]. DOCK180, which acts predominantly on the Rho family GTPase Rac1, restored cell locomotion in cells expressing Phe-31/118 paxillin mutants deficient in Rac1 GTP-loading, suggesting that formation of paxillin-Crk-DOCK180 signaling complex controls collagen-dependent migration mainly through Rac1 activation. We have found that DOCK180 together with paxillin-Crk constitutes the main pathway transducing collagen-mediated signals for the activation of Rac1 during NBT-II cell migration.
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