ASAP3 Is a Focal Adhesion-associated Arf GAP That Functions in Cell Migration and Invasion*

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.

ASAP proteins are a subtype of Arf GAPs that have been implicated in oncogenesis. Three ASAP-type proteins have been identified so far. ASAP1 and ASAP2, but not ASAP3, contain an SH3 domain at the extreme C terminus (35,36). The gene for ASAP1 is amplified in uveal melanoma and expression levels of ASAP1 have been found to correlate with invasive potential in uveal melanoma and mammary carcinoma (34,37). In addition, ASAP1 associates with and regulates FAs, CDRs, and invadopodia/podosomes (26 -28, 34, 38), structures involved in cell migration and invasion (2, 3, 39 -41). Reducing ASAP1 expression has been reported to slow cell migration in some studies (25,34), accelerate cell migration in others (26,33), and decrease cell invasion (34).
ASAP3, previously called up-regulated in liver cancer 1 (UPLC1), DDEFL1 and ACAP4 (42,43), was identified in analyses of expression profiles of clinical hepatocellular carcinomas using cDNA microarray. The relative expression of ASAP3 was high in hepatocellular carcinoma (43). The expression level of ASAP3 correlated with cell proliferation and migration (42).
However, the localization of endogenous protein and the precise site of action have not been examined.
Here we characterize ASAP3 and test the hypothesis that ASAP3 and ASAP1, with highly similar structures, have redundant cellular functions in controlling cell migration and invasion. Biochemically, ASAP3 is similar to ASAP1; however, examination of the cellular function of ASAP3 revealed differences from ASAP1. First, ASAP3 localized in FAs and CDRs but, different from ASAP1, ASAP3 was not found in invadopodia or podosomes, structures reported to mediate invasion of cancer cells. Second, reduction of ASAP3 expression did not prevent the formation of invadopodia or podosomes; in contrast, reduction of ASAP1 blocked the formation of invadopodia and podosomes (34,38). Decreasing the expression level of ASAP3 reduced actin stress fibers. Third, reduction of ASAP3 expression levels slowed cell migration and invasion, whereas reduction of ASAP1 expression did not affect these activities. Based on our results, we conclude that ASAP3 and ASAP1 do not function redundantly but have distinct functions. At least for the  mammary carcinoma cell line we examined, ASAP3 contributes to invasion but ASAP1 does not. Given the critical role of ASAP1 for invadopodia formation (38), these results also support the conclusion that invadopodia are dispensable for invasion.
siRNA Preparation and Transfection-For RNA interference assays, 19-nucleotide small interfering RNA duplexes (siRNAs) with 3Ј-UU overhangs specific for the 3Ј-untranslated region (5Ј-3Ј) and the open reading frame ("Smart pool," combination of 4 siRNA duplexes) for human ASAP3/DDEFL1 (GenBank TM accession number NM_017707) were synthesized by Dharma-   MAY 30, 2008 • VOLUME 283 • NUMBER 22 Cell Culture and Cell Transfection-Human cell lines HepG2, U118, and MDA-MB-231 and mouse NIH 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO 2 atmosphere at constant humidity.

ASAP3 Controls Cell Invasion
Protein Expression and Purification from Bacteria-To express different constructs of ASAP3, reading frames for proteins of residues 287-693 (PZA), 1-693 (BAR-PZA), 417-903 (ZA-proline rich), 293-903 (⌬BAR ASAP3), and 1-903 (full-length ASAP3) of ASAP3 were ligated into the NdeI/XhoI sites of pET19b (Novagen), which contains a His 10 fusion at the N terminus to express histidine-tagged proteins. Recombinant proteins were expressed in and purified from Escherichia coli over a HiTrap Q anion-exchange column (GE Healthcare) followed by a nickel affinity column (GE Healthcare) using gradients of NaCl and imidazole. Experiments were performed with the histidine-tagged proteins. Non-myristoylated Arfs were prepared as described (44).
Immunofluorescence-Cultured cells were seeded on fibronectin-coated coverslips for at least 4 h and fixed in 2% formaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature, washed three times with phosphate-buffered saline (PBS), and once in 10% fetal bovine serum and 0.04% sodium azide in PBS. Cells were then permeabilized in buffer A (10% fetal bovine serum, 0.04% sodium azide, and 0.2% saponin in PBS) for 10 min at room temperature. Cells were incubated with primary antibodies at 1:100 dilution in buffer A for 1 h at room temperature followed by incubation with appropriate secondary antibodies conjugated with fluorescein isothiocyanate (Jackson ImmunoResearch) for 1 h at room temperature. For visualization of actin filaments, rhodamine-conjugated phalloidin (Molecular Probes) was added during the incubation with secondary antibodies. Coverslips were then washed and mounted on glass slides with Gel Mount (Biomeda). Cells were visualized and images captured by confocal microscopy using a Zeiss LSM 510 attached to a Zeiss Axiovert 100M with a 63 ϫ 1.4 NA plan Neofluar oil immersion lens. Adobe Illustrator was used to prepare the images for publication. Western Blot Analysis-Cell lysates containing equivalents of total protein were resolved on a 10% SDS-polyacrylamide gel, followed by transfer to nitrocellulose membranes (Bio-Rad). Membranes were incubated in 4% nonfat dry milk for 1 h at room temperature, washed with PBS containing 0.1% Tween 20, followed by incubation with the primary antibody (as specified) for 1 h at room temperature or overnight at 4°C. Membranes were then washed and incubated in the appropriate secondary antibody for 1 h at room temperature, and the immunocomplexes were visualized using ECL Plus Western blotting Detection System (Amersham Biosciences).
Cell Proliferation Assay-Cells treated with siRNA targeting ASAP3 or cells stably expressing ASAP3 or [R469K]ASAP3 were plated at a density of 20,000 cells per well in 12-well plates in 1 ml of culture medium containing 10% fetal bovine serum. Proliferation was monitored by counting cells each day using a hemocytometer. The experiments were repeated twice in triplicates each.
MTT Assay-Cells previously treated with the indicated siRNA were plated at a density of 3,000 cells per well in 96-well plates (200 l/well) and incubated at 37°C. MTT assay was performed every 24 h by adding 20 l of MTT (5 mg/ml) 4 h before cell harvesting. The resulting MTT purple formazan crystals in each well were solubilized by addition of 100 l of DMSO and absorbance was measured spectrophotometrically at a wavelength of 650 nm.
Wound Healing Assay-Monolayers of confluent cells were scratched with a sterile pipette tip and phasecontrast images of cells were taken either immediately after wounding or after 24 or 48 h. Repair of the artificial "wound" (% healing) was quantified as follows: healing (%) ϭ ((1 Ϫ (width of wound at 0 h/width of wound at 24 or 48 h)) ϫ 100.
Transwell Migration and Invasion Assay-Migration and invasion assays were carried out with cell cul- ture inserts (8-m pore size; BD Biosciences). For invasion assays, cell culture inserts were coated with 10 g of Matrigel (BD Biosciences). The Matrigel was diluted in cold serum-free medium and added to the chambers and dried in a sterile hood for 4 -5 h. The Matrigel was then reconstituted with serum-free medium for 1 h at 37°C before the addition of cells.
Cells were harvested by trypsinization, resuspended in Opti-MEM and 1 ϫ 10 4 cells were added to the upper chambers, and the chambers were placed in 24-well dishes. The lower compartment of the wells were filled with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum used as chemoattractant. Migration assays were carried out for 12 h and invasion assays for 24 h. Cells were scraped off the top side of the polyethylene membrane with a cotton swab. Cells that invaded into the Matrigel and migrated out onto the lower surface of the membrane were stained in 0.2% crystal violet. Images of the cells that invaded the lower surface of the filter were taken with the ϫ2.5 objective of an inverted microscope (Nikon Eclipse E800) coupled to a digital camera (Hamamatsu C4742-95). Data were collected from at least three independent experiments each carried out in triplicate. Data are presented as percentages calculated by normalizing the values obtained for the cells treated with a negative control siRNA as 100%.
Site-directed Mutagenesis-Mutations were performed using the QuikChange kit (Stratagene) following the manufacturer's instructions and confirmed by DNA sequencing. Mutant proteins were purified as described above.
Arf GAP Assay-Arf GAP activity was determined using an in vitro assay that measures a single round of GTP hydrolysis on recombinant Arf (45,46

ASAP3 Controls Cell Invasion
presence of large unilamellar vesicles (LUVs). Reactions were terminated after 3 min by adding ice-cold buffer. Proteinbound nucleotide was trapped on nitrocellulose filters, released into formic acid, fractionated by chromatography on polyethyleneimide cellulose plates, and quantified using a Phosphor-Imager (GE Healthcare). All experiments were performed at least three times with similar results.
Statistics-Numerical data are presented as the mean Ϯ S.E. The data were analyzed by analysis of variance, followed by Dunnett's test to determine the statistical significance of differences. All statistical analyses were performed using GraphPad Prism for Windows version 4.0 (GraphPad).

Nomenclature
The protein we examine here was first called UPLC1. The protein was subsequently called development and differentiation enhancing factor like-1 (DDEFL1) because of similarity to ASAP1/DDEF1 (43) and ACAP4, because of similarity to ACAP subtype Arf GAPs (42). Computational analysis of the amino acid sequence of UPLC1/DDEFL1 identified common reported motifs such as the PH, Arf GAP, and ankyrin repeat domains (Fig. 1A). To determine the most appropriate name for the protein, we analyzed the primary sequence and phylogeny of the highly conserved Arf GAP domain of the reported mammalian Arf GAPs (Fig. 1, B and C). By both analyses, the protein first called UPLC1 is an ASAP-type protein and we, therefore, will refer to the protein as ASAP3. The deduced 903-amino acid sequence of ASAP3 showed 50% similarity with ASAP1 based on the analysis of the primary structure of the proteins.
The dependence of GAP activity on the PH domain was also determined. [417-903]ASAP3, which lacks the PH domain, was compared with [293-903]ASAP3 in GAP assays (Fig. 4)

Cellular Function of ASAP3
Localization-An antibody, raised, purified, and characterized as described under supplemental Materials, was used to  MAY

ASAP3 Controls Cell Invasion
determine the localization of ASAP3. ASAP3 was found to colocalize with two markers of FAs, vinculin and paxillin, in MDA-MB-231 (breast cancer) and U118 (glioblastoma) cells (Fig. 5A). Similarly, epitope-tagged ASAP3, expressed in MDA-MB-231 cells, associated with FAs (Fig. 5B). We next determined whether ASAP3 also associated with CDRs and invadopodia. To visualize CDRs, NIH 3T3 fibroblasts, which stably expressed FLAG-ASAP3, were treated with platelet-derived growth factor. After 5 min, CDRs were detected as rings of polymerized actin on the dorsal surface of the cells. ASAP3 was found in the CDRs (Fig. 6). To visualize invadopodia in MDA-MB-231 cells and podosomes in NIH 3T3 fibroblasts, cells were transfected with plasmids expressing the active form of Src ([527F]c-Src). Under this condition, invadopodia and podosomes were detected, using actin or cortactin as markers, as punctate or ring-like structures on the ventral surface of the cell (Fig. 7). Endogenous ASAP3 (Fig. 7A) was not detected in either structure. Recombinant ASAP3 was also examined in MDA-MB-231 cells (Fig. 7B). It did not localize to invadopodia.
Effects of ASAP3 on the Cytoskeleton-We considered the possibility that ASAP3 affects the structure and function of FAs. As initial tests, recombinant ASAP3 was expressed to high levels in MDA-MB-231 cells. FAs were visualized with antibodies to paxillin and vinculin (Fig. 8A). In contrast to ASAP1 that causes dissociation of paxillin (26,33), ASAP3 had no effect (Fig. 8C). [R469A]ASAP3, which does not have detectable GAP activity (data not shown), also did not affect the distribution of vinculin or paxillin (Fig. 8D). We also examined the effect of reducing ASAP3 expression, through the use of siRNA (Fig. 8, B and E). The protein level was reduced by 90% but no effect on the distribution of paxillin or vinculin was observed. We concluded that ASAP3 does not affect FAs under these conditions. The effect of reduced ASAP3 expression on other cytoskeletal structures was examined. Invadopodia formation was not affected by reducing ASAP3 levels (Fig. 9). In the experiments examining focal adhesions (Fig. 8) and invadopodia (Fig. 9), polymerized actin was visualized with rhodamine-phalloidin. We noted that cells with reduced ASAP3 protein levels showed a disruption of the actin-containing stress fiber network compared with control cells (Figs. 8, B and E, 9, and 10). The effect  was independent of the siRNA used to reduce ASAP3. Stress fibers in MDA-MB-231 cells could be partly restored by expressing ASAP3 from a plasmid that was resistant to the siRNA-mediated silencing of ASAP3 (Fig. 10A). Myosin phosphorylated on threonine 18 and serine 19 associates with and stabilizes actin stress fibers (4). We determined whether reduction of ASAP3 expression also affects phosphomyosin levels. In these experiments we examined both MDA-MB-231 and U118 cells. In both cell types, reduction of ASAP3 with siRNA resulted in decreased levels of myosin phosphorylated on serine 19 (Fig. 10, B-D). Phosphomyosin levels were restored if endogenous protein were replaced with recombinant ASAP3 by expression using an siRNA-resistant plasmid in U118 cells (Fig. 10, C and D). Taken together, our results support the conclusion that ASAP3 does not affect the structure of FAs but alters actin stress fibers that are associated with FAs.
Effects of ASAP3 on Cell Migration-We next examined whether ASAP3 affects cell migration. Treatment of MDA-MB-231 cells by ASAP3 siRNA suppressed the ASAP3 protein level by over 90% (Fig. 11A). Cell migration was examined in two ways. In a wound healing assay (Fig. 11, B and C), a monolayer of cells was scratched with a pipette tip, scraping cells from the surface. The movement of the cells into the artificial wound was observed over the course of 48 h. ASAP1 has previously been shown to affect cell migration (25,26,33) and was used as a control. We found that MDA-MB-231 with reduced ASAP1 migrated into the wound at the same rate as cells treated with an irrelevant siRNA. In contrast, reducing expression of ASAP3 reduced cell migration. Similar results were obtained using siRNA targeting a different sequence in the ASAP3 message.
Cell migration was also assessed using a transwell assay (Fig. 11D). As with the wound healing assay, MDA-MB-231 cells with reduced expression of ASAP3 migrated less than control cells; in contrast, reducing ASAP1 expression had no effect. Cell invasion involves migration through an extracellular matrix. To determine whether ASAP3 contributed to invasion, we used a MDA-MB-231 mammary carcinoma cells as a model and migration through Matrigel as an assay. Cells treated with siRNA targeting ASAP3 invaded less than cells treated with an irrelevant siRNA or untreated cells. The effect was independent of the particular targeting siRNA used. Although ASAP1 has been reported to be required for the formation of invadopodia (34,38) and to be required for invasion (34), we found that the reduction of ASAP1 expression had no effect on invasion through Matrigel (Fig. 11E). Role of ASAP3 in Cell Proliferation-Increased expression levels of ASAP3 have been reported to accelerate the proliferation of NIH 3T3 fibroblasts (44). To determine whether ASAP3 affects proliferation of other cell types, MDA-MB-231 cells were treated with siRNA targeting ASAP3 or an irrelevant RNA and cultured up to 7 days. In one assay, cell mass was determined with the MTT reagent. No difference between the cells treated with siRNA targeting ASAP3 and those treated with irrelevant RNA was observed (Fig. 12A). As a second measure of cell proliferation, cell number was determined using a hemocytometer. No differences in growth rates were observed (Fig.  12B). In a similar experimental design, NIH 3T3 fibroblasts stably expressing ASAP3 or [R469K]ASAP3 were seeded at subconfluent levels and grown for 7 days. No differences in the rate of cell proliferation were observed (Fig. 12C). Taken together, our data suggest that ASAP3 does not promote cell proliferation.

DISCUSSION
ASAP3 was first identified as a protein that contributes to cell proliferation in hepatocellular carcinoma and was later reported to be an Arf6-specific Arf GAP that regulates cell migration (42,43). We set out to test whether ASAP3 functions redundantly with an Arf GAP of the same class, ASAP1, which has been implicated in the same processes. We found that, like ASAP1, ASAP3 associates with FAs and CDRs. However, ASAP3 was different from ASAP1 in two respects. First, ASAP3 was not found in nor was required for the formation of invadopodia. Second, reduction of ASAP3 levels reduced both cell migration and invasion rates, whereas reduction of ASAP1 had no detectable effect on invasion or migration. We conclude on the basis of these results that ASAP1 and ASAP3 function nonredundantly in the control of cell movement.
One unexpected result of this work was that reduction of ASAP1 expression did not slow cell invasion, whereas reduction in ASAP3 did. Cells with reduced ASAP1 expression do not form invadopodia (38,47), which are thought to mediate invasion. It is possible that the cells formed invadopodia-like structures with equivalent function, which we did not detect. Nevertheless, these results support the idea that invadopodia, as currently defined, are not necessary for invasion through Matrigel. Cells with reduced ASAP3 did form invadopodia, but did not invade efficiently. Thus, invadopodia are not sufficient for invasion.
Cells with reduced ASAP3 expression had less filamentous actin, consistent with current paradigms that ascribe a role for actin filaments in cell movement (48 -52). With the decrease in actin stress fibers, there was less phosphomyosin. ASAP3 is found in FAs, which are complexes of proteins that attach to actin stress fibers. FAs and actin stress fibers are coordinately regulated, but some recent evidence supports the idea that they can be individually controlled. ARAP2, another Arf GAP found in FAs appears to be a Rho effector that regulates the formation of FAs (19). Reduction of ARAP2 expression blocks FA and stress fiber formation. Stress fibers, but not FAs, can be recovered by expression of activated Rho kinase. Thus, ARAP2 appears to have a greater influence on FA than stress fibers. Here we found that reduction of ASAP3 does not affect FA formation, but does disrupt filamentous actin. Based on these results, we conclude that Arf GAPs with a common localization could have independent functions. Ongoing work is focused on defining the molecular basis for cytoskeletal effects specific to different FA-associated Arf GAPs.
Arf GAPs can affect actin polymerization by a number of mechanisms (10), which may be relevant to ASAP3. For instance, Arf GAPs can interface with the Rho pathway. One class of Arf GAPs, called Gits, bind to PIX, a Rac/Cdc42 exchange factor, and p21 activated kinase (PAK), a Rac effector (22,31,32,53). Another class of Arf GAPs, the ARAPs, contain Rho GAP domains (19, 54 -58) and affect actin by converting RhoA⅐GTP to RhoA⅐GDP. Arf GAPs may also affect actin by regulating Arf proteins. Arf6, for instance, has been found to regulate a Rac exchange factor (59) and the trafficking of Rac1 (60,61). Arf6 has also been reported to bind directly to Rac1 (62). Identification of associating proteins may reveal if any of these mechanisms apply to ASAP3.
The differential effects of ASAP1 and ASAP3 could be consequent to differences in Arf specificity. The Arf specificity of ASAP1 has not been clearly established. Several lines of evidence support the conclusion that ASAP1 is an Arf1 or Arf5 GAP (25,26,33,35) but ASAP1 has also been reported to use Arf6 as a substrate (9). Our in vitro experiments indicated ASAP3 has a small preference for Arf5 and Arf1 as compared with Arf6. However, our in vivo studies were not conclusive (not shown). Previous reports supported the idea that Arf6 is the preferred substrate in vitro; however, in that work, GSTtagged Arf was used as a substrate (42). Given that GST is 26 kDa and Arf has a mass of 20 kDa, the GST could have affected the structure of Arf. Based on prevailing models for Arf action in cell migration, Arf6 is predicted to be the substrate. Examin-ing the possibility that the ASAPs differ in specificity for class 1 and class 2 Arfs will require an expanded analysis and development of new experimental strategies.
In contrast to previous work, we did not observe an effect of ASAP3 on cell proliferation. We used three assays. First, we used the MTT assay as a means to measuring changes in total cell mass. Second, growth curves were determined. We also examined thymidine incorporation that did not reveal effects of ASAP3 (data not shown). The previously observed effects were modest (43). We may have not detected the effect due to lack of sensitivity in our assays. In summary, ASAP3 was identified as an Arf GAP that may regulate cell migration associated with the invasion of normal tissue by cancer cells.