Sonja Krugmann

ARAP3 Is a PI3K- and Rap-Regulated GAP for RhoA

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3s Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.

P-Rex2, a new guanine-nucleotide exchange factor for Rac

We have identified a new guanine‐nucleotide exchange factor, P‐Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P‐Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)‐trisphosphate and the βγ subunits of heterotrimeric G proteins in vitro and in vivo. P‐Rex2 has structure, activity and regulatory properties similar to P‐Rex1 but has divergent tissue distribution, as P‐Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein‐coupled receptors and phosphoinositide 3‐kinase.

ARAP3 is essential for formation of lamellipodia after growth factor stimulation.

Rho and Arf family small GTPases control dynamic actin rearrangements and vesicular trafficking events. ARAP3 is a dual GAP for RhoA and Arf6 that is regulated by phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3], a product of the phosphoinositide 3-kinase (PI3K) signalling pathway. To investigate the physiological function of ARAP3, we used an RNAi-based approach in an endothelial cell model. ARAP3-deficient cells showed increased activities of RhoA and Arf6. Phenotypically, they were more rounded than control counterparts and displayed very fine stress fibres. ARAP3-deficient cells were not capable of producing lamellipodia upon growth factor stimulation, a process known to depend on PI3K and Rac activities. Rac was transiently activated in stimulated ARAP3 RNAi cells although its cellular localisation was altered, a likely consequence of increased Arf6 activity. We conclude that ARAP3 recruitment to sites of elevated PtdIns(3,4,5)P3 is crucial to allow localised inactivation of RhoA and cycling of Arf6, both of which are necessary to allow growth factor-stimulated formation of lamellipodia.

Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein betagamma subunits.

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein betagamma subunits (Gbetagammas). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P(2), and the Gbetagammas are localized at the plasma membrane. We have addressed the mechanism by which Gbetagammas regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gbetagammas incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gbetagammas. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gbetagammas in vitro. We conclude that Gbetagammas activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

Purification of ARAP3 and Characterization of GAP Activities

ARAP3 is a dual Arf and Rho GTPase activating protein (GAP) that was identified from pig leukocyte cytosol using a phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns[3,4,5]P3) affinity matrix in a targeted proteomics study. ARAP3s domain structure includes five PH domains, an Arf GAP domain, three ankyrin repeats, a Rho GAP domain, and a Ras association domain. ARAP3 is a PtdIns(3,4,5)P3-dependent GAP for Arf6 both in vitro and in vivo. It acts as a Rap-GTP-activated RhoA GAP in vitro, and this activation depends on a direct interaction between ARAP3 and Rap-GTP; in vivo PtdIns(3,4,5)P3 seems to be required to allow ARAP3s activation as a RhoA GAP by Rap-GTP. Overexpression of ARAP3 in pig aortic endothelial (PAE) cells causes the PI3K-dependent loss of adhesion to the substratum and interferes with lamellipodium formation. This overexpression phenotype depends on ARAP3s intact abilities to bind PtdIns(3,4,5)P3, to interact with Rap-GTP, and to be a catalytically active RhoA and Arf6 GAP.

The roles of PI3Ks in cellular regulation

The term phosphoinositide 30H kinase (PI3K) is given to a family of enzymes which can phosphorylate one or more of the conventional inositol phospholipids found in cells in the 3-position of their inositol headgroup (Fig. 1). It is now clear that these lipids act as regulators of intracellular metabolism and at least one of them, PtdIns(3,4,5)P3, shows all the credentials of being a major ‘second-messenger’ in signalling pathways used by cell-surface receptors for growth factors, inflammatory stimuli and antigens (Stephens et al., 1993; Cantley et al., 1991).