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 ␤ family, ASAP1 (also known as centaurin ␤4), 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.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2cko/ko mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2ko/ko cells. PARP1 deficiency does not restore HR in Brca2ko/ko cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2cko/cko mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin ␤ family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin ␤4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P 2 ) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P 2 binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P 2 binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf. Arf1 proteins are ubiquitous and essential GTP-binding proteins in eukaryotes. They were first identified as cofactors for cholera toxin-catalyzed ADP-ribosylation of G s (1). The relationship between this activity and the normal cellular function of Arf is not clear. The best described activity of Arf is the regulation of membrane traffic (2, 3). More recently, potential roles in reorganization of the actin cytoskeleton have been identified for Arf1 and Arf6 (4 -7). Neither the molecular mechanism by which Arf proteins regulate these pathways nor the means by which Arf itself is regulated have been delineated; however, phospholipids probably function in both capacities.The relationship between Arf and phospholipids is complex. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase, resulting in the production of phosphatidic acid (PA) and PtdIns-4,5-P 2 (8 -11). Phosphoinositides and phosphatidic acid also regulate Arf function. Arf itself binds PtdIns-P 2 (12). Phosphoinositides have a role in converting Arf-GTP to Arf-GDP by the centaurin ␤ family of Arf GAPs. Four members of this family have been identified 2 (13, 14), and one member of the family, ASAP1 (also called centaurin ␤4), was purified as an Arf GAP that is coordinately activated by PA and phosphoinositides (13). The Arf⅐PtdIns-P 2 complex was found to be the substrat...