Abstract
Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)-positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)-dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.
Highlights
Focal adhesion kinase (FAK) is a central regulator of integrindependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins
Because FAK is a central effector of downstream integrin engagement [1] and because Rab5 stimulates cellular responses that are reminiscent of those initiated by FAK, such as focal adhesions (FAs) assembly and disassembly [9], Rac1 activation [14, 15], persistent cell migration [9, 11], and matrix metalloproteinase activation [9, 23], we hypothesized that FAK is an upstream regulator of Rab5 that promotes GTP loading via an intermediate regulator yet to be identified
A549 cells, which are known to adhere to fibronectin via integrin ␣51 [9, 13], were treated with the small molecule PF562,271, which inhibits FAK autophosphorylation on Tyr397 [24], and Rab5-GTP levels were measured by pulldown, as reported previously [9, 18]
Summary
X Cecilia Arriagada‡, Patricio Silva‡§, Martial Millet‡, Luis Solano‡, Carolina Moraga‡, and X Vicente A. Torres‡¶1 From the ‡Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago 8380000, Chile, the §Faculty of Health Sciences, Universidad Central de Chile, Santiago 8380000, Chile, and the ¶Advanced Center for Chronic Diseases, Universidad de Chile, Santiago 8380000, Chile
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.