Abstract
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.
Highlights
Cell migration is important to physiological processes such as development and inflammation and disease processes such as the invasion of normal tissue by cancer cells
In comparing ASAP1 and ASAP3, we found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma
The Arf GTPase-activating proteins (GAPs) are a family of multidomain proteins with the common catalytic function of accelerating the hydrolysis of GTP bound to Arf (8, 10, 20 –22), thereby inactivating Arf proteins
Summary
ASAP proteins are a subtype of Arf GAPs that have been implicated in oncogenesis. Three ASAP-type proteins have been identified so far. 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]. 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). 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. 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. Reduction of ASAP3 expression levels slowed cell migration and invasion, whereas reduction of ASAP1 expression did not affect these activities. Given the critical role of ASAP1 for invadopodia formation [38], these results support the conclusion that invadopodia are dispensable for invasion
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