Phosphatidylinositol 4-Phosphate 5-Kinase α Is a Downstream Effector of the Small G Protein ARF6 in Membrane Ruffle Formation

Abstract

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…Similarly, Arf6 stimulates recruitment and activation of type I PIP5 kinases at the plasma membrane and endosomes [6,7], a property shared with its yeast counterpart, yArf3 [8]. At both the Golgi and the plasma membrane, the Arf-dependent recruitment of PtdIns kinases results in enhanced local production of PtdIns(4,5)P 2 .…”

“…and neurite outgrowth [2]. In a similar fashion to Arf1, Arf6 can activate both phospholipase D and type I PIP5 kinases, but it does so at the plasma membrane and on endosomal compartments [6]. Consistent with this function, expression of the Arf6-specific GEF, EFA6 (exchange factor for Arf6), or a constitutively active Arf6 mutant, Arf6Q67L, leads to the formation of swollen, clustered endosomes that have membranes enriched in PtdIns (4,5)P 2 and surrounded by polymerized actin [25].…”

Arf6 regulates tumour angiogenesis and growth through HGF-induced endothelial β1 integrin recycling

“…Moreover, the GTP-bound active form of Arf6 interacts with Sec10, a subunit of the exocyst complex involved in vesicle targeting to and tethering with the plasma membrane 48 . In addition, the well-known Arf6 effector phosphatidylinositol 4-phosphate 5-kinase 18 , which produces the versatile signalling lipid phosphatidylinositol 4,5-bisphosphate, regulates tethering of vesicles containing b1 integrin to the plasma membrane and/or fusion with it at the leading edge of migrating cells through the interaction of generated phosphatidylinositol 4,5-bisphosphate with the exocyst protein Exo70 (ref. 49).…”

“…The small GTPase Arf6 plays important roles in cell migration and membrane dynamics by regulating intracellular membrane trafficking and reorganizing the actin cytoskeleton 17,18 . Arf6 cycles through a GDP-bound inactive and a GTP-bound active form.…”

Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines

“…The activation of ERM proteins invariably requires PIP2 local accumulation (Hartwig et al 1995;Tolias et al 2000;Honda et al 1999). Two putative binding sites were identified, and PIP2 binding was demonstrated to be essential in guiding ezrin to cell surface locations (Barret et al 2000).…”