The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton

Abstract

Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin beta family, ASAP1 (also known as centaurin beta4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.

Figure 1

ASAP1 associates with FAs. Arrows point to FAs. (A) Antibody specific for ASAP1. Antiserum 642 was used for Western blotting (Left) at a 1:10,000 dilution and for immunofluorescence (Right) at a dilution of 1:2,000. Cell lysate (1 μg) was used for Western blotting. The peptide (500 μM) to which the antibody was raised was included where indicated. (B) Colocalization of ASAP1 with markers of FAs. PY, protein phosphotyrosine. (Bars = 10 μm.)

Figure 2

ASAP1 cycles between FAs, the cytoplasm, and plasma membrane when cells are stimulated to move. (A) Cell spreading. NIH 3T3 fibroblasts were removed from plates with trypsin/EDTA and incubated in serum-free medium with fibronectin-coated glass coverslips for the indicated times. (Bars = 10 μm.) (B) PDGF-induced cytoskeletal remodeling: time course. PDGF (10 ng/ml) was added to cells on fibronectin-coated coverslips in serum-free medium. (C) PDGF-induced dorsal ruffles contain actin and vinculin. Cells were treated for 5 min with 10 ng/ml PDGF. Actin was visualized by using phalloidin conjugated to rhodamine. Arrows, dorsal ruffles; arrowheads, FAs.

Figure 3

Overexpression of ASAP1 affects cytoskeletal remodeling. NIH 3T3 fibroblasts were transfected with plasmids for FLAG-tagged ASAP1. (A) FA morphology. For double staining for β1 integrin, ASAP1 was detected by using a monoclonal antibody to the FLAG epitope. For paxillin, ASAP1 was detected with the polyclonal antibody 642. In this case, endogenous ASAP1 was apparent in FAs in nontransfected cells, visible in photographs in which the transfected cells were overexposed. (Bar = 10 μm.) (B) Comparison of transfected and nontransfected cells during spreading. A low-power field photographed from experiments used to derive the data for C is shown. (Bar = 40 μm.) At higher magnification, two transfected cells, fixed 1 and 2 h after plating, are shown. (C) Effect of ASAP1 overexpression on cell spreading rate. NIH 3T3 fibroblasts were seeded on fibronectin-coated coverslips. At the indicated times, cells were removed and fixed. Transfected cells were detected by using a monoclonal antibody to the FLAG epitope and were counterstained with rhodamine-phalloidin. A cell was considered spread when the total diameter was 2-fold or greater than the nuclear diameter. The data are the means and range for two experiments. (D) Effect of ASAP1 and [R497K]ASAP1 overexpression on cell spreading and PDGF-induced dorsal ruffles. Cells were transfected with plasmids encoding FLAG epitope-tagged ASAP1 or [R497K]ASAP1. Spreading was determined at 15 min. To examine ruffling, cells were treated for 4 min with 10 ng/ml PDGF. Dorsal ruffles were visualized by using rhodamine-phalloidin. Data are shown as mean ± SD for four experiments. ∗, Different from control, P < 0.001; +, different from wild type, P < 0.01. (E) Expression levels of FLAG-tagged ASAP1 and FLAG-tagged [R497K]ASAP1. Cells were transfected in parallel to those used for spreading and ruffling assay and lysed in RIPA buffer (0.15 mM NaCl/0.05 mM Tris⋅HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS). ASAP1 in 1 μg of cell lysate protein was detected with an antibody to the FLAG epitope (Left) and in 0.1 μg of cell lysate protein with antibody 642 (Right).