The role of Arf-GAP ASAP1 in integrin signaling.

摘要

A growing amount of evidence has implicated a prominent role for focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase localized to focal adhesions, in integrin signaling. The C-terminal region of FAK contains multiple protein-protein interaction modules which serve as the docking sites for a number of SH3 domain-containing proteins. The recruitment of these downstream effectors is believed to be important for FAK function.; To better understand the role of FAK in integrin signaling we attempted to identify proteins that interact with its C-terminal region using a yeast two-hybrid system. This has led to the identification of a novel FAK binding partner ASAP1, a GTPase-activating protein (GAP) for ADP ribosylation factor (Arf) GTPases.{09}ASAP1 binds to FAK both in vitro and in vivo. ASAP1 interacts with the second proline-rich motif of FAK through its C-terminal SH3 domain. Furthermore, ASAP1 co-localizes with FAK in focal adhesions. Overexpression of ASAP1 inhibited cell spreading on fibronectin and paxillin localization to adhesions and these inhibitory effects are dependent upon its interaction with FAK and its GAP activity.{09}Using proteomics approach we identified an ASAP1 binding partner CD2-associated protein (CD2AP) which interacts with ASAP1 through its N-terminal SH3 domains. Mis-localization of endogenous ASAP1 to mitochondria with CD2AP SH3 domains fused to a mitochondria-targeting sequence or attenuation of ASAP1 expression with siRNAs caused inhibition of cell spreading and migration in response to fibronectin stimulation. Furthermore, abrogation of ASAP1 function with either RNA interference or mis-localization approaches resulted in an increase of GTP loading on Arf1 and loss of paxillin from adhesions. Taken together, these results suggest that the recruitment of certain adhesion components such as paxillin may require dynamic turnover of Arf1 GTPase rather than its GTP-bound active form.; Through the characterization of FAK-interacting Arf-GAP ASAP1 we have gained evidences that implicate Arf GTPases in integrin signaling. The observations described in this dissertation along with future efforts in this field will provide insight into the role of Arf-GAPs in integrin signaling and a more comprehensive view of FAK function.