ACAPs are arf6 GTPase-activating proteins that function in the cell periphery

Abstract

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

Figure 1

Structure of ACAP1 and ACAP2. (A) Comparison of primary sequence and structural domains. The sequence of ACAP1 and ACAP2 were aligned using the program GAP in GCG. Structural domains were identified using Pfam, ProfileScan, and COILS. Sequence that is shaded gray is homologous to oligophrenin-1. Sequence comprising coiled coil, PH, Arf GAP, and ANK-repeat domains is indicated by green, red, bold italic, and dark blue text, respectively. Homology to the PI-PLC X-box is indicated by light blue text. (B) Schematic of ASAP/ACAP family members. The “X Box” refers to the PI-PLC X-box. “Pro” refers to a proline-rich domain with SH3-binding sites. “E/DLPPKP” is a domain of tandem repeats of this consensus sequence. (C) Comparison of ASAP/ACAP family protein PH domains. The sequences were aligned using ClustalW. Identities between ACAP1 and ACAP2 are indicated in yellow, between PAP and ASAP1 in blue, and common identities in green. (D) Comparison of ACAP1 and ACAP2 to oligophrenin-1. The sequences were aligned using ClustalW.

Figure 2

Expression of ACAP1 and ACAP2. (A) Tissue distribution. Message levels in human tissues were compared by semiquantitative PCR, as described in Materials and Methods. Message for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as a control. (B) ESTs encoding ACAPs. The sequence of ACAP1 and ACAP2 were used to perform a BLAST search of the EST database. Nucleotide sequence encoding identical proteins are listed with tissue source and accession no. (C) Comparison of relative protein levels in cultured cell lines. ACAP1, ACAP2, and ASAP1 were detected by Western blot using antibodies described in Materials and Methods. Arf6 and tubulin were used as controls for normalization.

Figure 5

A conserved arginine in ACAP/ASAP family members is necessary for GAP activity. epitope-tagged ACAP1, [R448Q]ACAP1, ACAP2, [R442Q]ACAP2, ASAP1, and [R497K]ASAP1 were expressed and purified as described in Materials and Methods. The GAP activity of ACAP1 and ACAP2, using Arf6 as a substrate, was compared with the GAP activity of an equal amount of [R448Q]ACAP1 and [R442Q]ACAP2. The GAP activity of ASAP1 and [R497K]ASAP1, present at equivalent concentrations, was determined using Arf1 as a substrate. The data were normalized to the activity of wild-type protein, which was 0.05/min for ACAP1, 0.088/min for ACAP2, and 0.35/min for ASAP1. The error bars represent the range of duplicate determinations from one of three experiments.

Figure 3

Arf specificity of ACAP GAP activity. Arf GAP assays were performed using epitope-tagged ACAP1, ACAP2, and ASAP1 expressed in HEK 293 cells and purified as described in Materials and Methods. Reactivity to Arf GAP epitope tags were used to quantify the proteins. Arf1, 5, and 6 were used in a reaction that included 90 μM PIP2, 380 μM PA, and either ACAP1, ACAP2, or ASAP1. Arf GAPs were added in quantities to give reaction rates between 0.025 and 0.25 hydrolyses/min. The data are normalized for the amount of GAP added. The data are representative of two experiments, and the error bars are the range of duplicates. Similar results were obtained using at least two separate preparations of each protein.

Figure 4

PIP2 and PA dependence of ACAP/ASAP family Arf GAPs. Arf6 was used as a substrate for ACAP1 and ACAP2. Arf1 was used as a substrate for 0.2 μg/ml ASAP1 purified from bovine brain. PIP2 concentrations are indicated. The reactions contained 380 μM PA, where indicated. The rates are given in hydrolyses/min.

Figure 6

Ectopically expressed ACAPs in PDGF-stimulated NIH 3T3 fibroblasts. NIH 3T3 cells expressing epitope-tagged ACAP1, [R448Q]ACAP1, ACAP2, and [R442Q]ACAP2 were treated for 4 min with PDGF, fixed, and immunostained. Actin was visualized with rhodamine–conjugated phalloidin. Ruffles are indicated by arrowheads. Bars, 10 μm.

Figure 7

ACAPS inhibit the formation of PDGF-induced dorsal actin–rich ruffles in NIH 3T3 fibroblasts. The fraction of cells forming dorsal ruffles in response to PDGF was determined in cells transfected with either empty vector or vectors directing expression of the indicated ACAPs, including point mutants, [R448Q]ACAP1 and[R442Q]ACAP2, that lack GAP activity. The data presented are the mean ± SEM of five experiments.

Figure 11

Effects of ACAPs are dependent on both the formation of Arf-GTP and GAP activity. Cells were transfected with either plasmids directing expression of the dominant-negative (T27N) form or the constitutively active (Q67L) form of Arf6 and ACAP1. Cells expressing Arf6 and [R448Q]ACAP1, which lacks GAP activity, were treated with AlF4 for 30 min before preparation for immunofluorescence. Bars, 10 μm.

Figure 8

Effect of ACAPs on Arf6-dependent protrusions and tubules in HeLa cells. Cells were transfected with plasmids directing expression of Arf6 and epitope-tagged ACAPs. 24 h later, cells were treated as indicated and prepared for immunofluorescence. Protrusions are indicated by arrowheads and membrane tubules by arrows. Bars, 10 μm.

Figure 9

Quantitation of the effect of ACAPs on Arf6-dependent formation of protrusions. Cells were transfected with Arf6, and the indicated ACAP/ASAP family Arf GAP was treated with AlF4 for 30 min and fixed. The data are presented as the fraction of cells forming protrusions when coexpressing the indicated Arf GAP, normalized to the fraction of cells expressing Arf6 alone, that formed protrusions. Means ± SEM for four experiments are presented.

Figure 10

Endogenous ACAP2 associates with Arf6-dependent tubes and protrusions. After a 30-min treatment with AlF4, HeLa cells expressing Arf6-HA were stained with an antibody for ACAP2. Bar, 10 μm.