The Association of ASAP1, an ADP Ribosylation Factor-GTPase Activating Protein, with Focal Adhesion Kinase Contributes to the Process of Focal Adhesion Assembly

Abstract

ASAP1 (ADP ribosylation factor [ARF]-GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src . Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK. INTRODUCTIONAttachment of cells to the extracellular matrix (ECM) is primarily mediated by the integrin family receptors (Hynes, 1992). Engagement of heterodimeric integrin receptors leads to the clustering of integrins and recruitment of numerous proteins to form multi-protein complexes on the cytoplasmic face of the plasma membrane termed focal adhesions (Burridge et al., 1988). Focal adhesions serve to anchor actin cytoskeleton to the plasma membrane and to provide a linkage between the extracellular environment and the cytoplasm (Burridge and Chrzanowska-Wodnicka, 1996). The recruitment of cytoskeletal proteins and the assembly of focal adhesions are functionally important for a number of cellular processes, including cell migration, survival, and proliferation (Lauffenburger and Horwitz, 1996). In the case of migrating cells (or cells spreading on ECM proteins), there is a requirement for the coordinated reorganization of the actin cytoskeleton and the formation of new attachments with the substratum (Huttenlocher et al., 1995;Bretscher, 1996). This process is temporally and spatially controlled, consistent with integrins functioning as both cell adhesion receptors and as initiators of signaling cascades that convey signals from ECM to actin cytoskeleton (Bretscher, 1996).Because integrins are catalytically inactive, their signaling ability is dependent upon the recruitment and activation of Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-01-0018. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-01-0018.* Corresponding a...

Src-family tyrosine kinase fyn phosphorylates phosphatidylinositol 3-kinase enhancer-activating Akt, preventing its apoptotic cleavage and promoting cell survival

“…PIKE-A possesses similar structural domains as ASAP1 (ArfGAP containing SH3, ankyrin repeats and PH domain), which prominently binds to both FAK and pyk2 (proline-rich tyrosine (y) kinase 2), a nonreceptor tyrosine kinase. 21,22 Unfortunately, PIKE-A does not bind to either tyrosine kinase (data not shown). ASAP1 interacts with FAK or pyk2 through its SH3 domain, however, PIKE-A does not contain any SH3 domain.…”

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

“…In preliminary experiments, we have observed that RhoA siRNA knockdown strongly decreases cell spreading in Caco-2 cells (data not shown), which would be consistent with a potential role for FAK inhibition of RhoA in regulation of cell spreading. The ADP ribosylation factorGTPase activating protein, ASAP1, also associates with FAK via the second SH3 domain and inhibits cell spreading on fibronectin when overexpressed in REF52 cells (Liu et al, 2002). However, the identification and characterization of these additional FAK-dependent signaling pathways in Caco-2 cells is beyond the scope of the present investigation.…”

“…In this system, the role of FAK in regulating p130 Cas function was not described. Additionally, in vivo observations of three-dimensional focal adhesions indicate the presence of tyrosine-phosphorylated paxillin, but not autophosphorylated FAK, in these structures (Cukierman et al, 2001), and other studies indicate that FAK interacts with proteins that negatively regulate cell spreading (Taylor et al, 1998(Taylor et al, , 1999Liu et al, 2002). In the present work, we utilized siRNAs to knock down protein levels of FAK and the related kinase Pyk2 in order to examine their role in regulation of p130 Cas function and in Caco-2 cell spreading on collagen IV.…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…ASAPs are phosphorylated by Src, FAK and Pyk2, and phosphorylation by Pyk2 has been shown to inhibit Arf GAP activity [58]. ASAP1 function appears to be important for focal adhesion dynamics, because both overexpression and knockdown have been shown to affect FA assembly [59,60]. However the precise role of ASAP1 within focal adhesions remains to be determined.…”