Actin Binding to the BAR Domain and Arf GAP Activity of ASAP1 Coordinately Control Actin Stress Fibers and Focal Adhesions

Abstract

Background

Actin stress fibers (SFs) and focal adhesions (FAs) are dynamic structures crucial to a range of cell behaviors including cell morphology, cell migration, proliferation, survival, and differentiation. The Arf GAP ASAP1 affects both SFs and FAs. Here, we test the hypothesis that two domains with distinct biochemical activities in ASAP1, the BAR domain that binds actin and nonmuscle myosin 2 (NM2) and the Arf GAP domain, which is necessary for inducing hydrolysis of GTP bound to Arf, coordinately regulate the structures.

Results

We found that ASAP1 associated with bundled actin, including SFs, colocalizing with α-actinin and nonmuscle myosin 2A (NM2A), and with paxillin in FAs. Reducing ASAP1 expression altered both SFs and FAs in four cell lines that we examined. The effects of reducing ASAP1 expression could be reversed by ectopic expression of ASAP1. Reduced expression of Arf5, a substrate for ASAP1, or expression of either dominant negative or GTPase deficient mutants of Arf5, affected SFs and FAs similarly to ASAP1 knockdown. Both an active GAP domain and a BAR domain contained in the same ASAP1 polypeptide were necessary to maintain FAs and SFs.

Conclusions and Significance

Taken together, the results support the idea that ASAP1 coordinates the maintenance of FAs and SFs through integrated function of the BAR and GAP domains. We speculate that ASAP1 regulates SFs and their interaction with FAs through direct binding to components of the actin cytoskeleton. We discuss hypotheses related to this Arf-dependent activity of ASAP1 and propose the function of ASAP1 is not control of Arf•GTP levels.