Type Iα phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly

Abstract

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.

Phosphoinositides in Constitutive Membrane Traffic

“…A major source of PA is through the hydrolysis of phosphatidylcholine by phospholipases D 1 and D 2 , enzymes that are themselves activated by PtdIns(4,5)P 2 (326). In vitro the PIP5KIs can phosphorylate phosphoinositides other than PtdIns(4)P and will convert PtdIns(3,4)P 2 to PtdIns(3,4,5)P 3 and PtdIns(3)P to both PtdIns(3,5)P 2 and PtdIns(3,4,5)P 3 (106,356,409).…”

Regulation of endocytic traffic by Rho GTPases

“…The type Iα PtdIns4P 5-kinase is also recruited to the phagocytic cup, and provides localized PtdIns(4,5)P # for regulation of F-actin [17]. This lipid kinase is directly activated by Rac [18]. Finally, the serine\threonine kinase PAK (p21-activated kinase), a downstream effector of both Rac and Cdc42 effector [19], is also targeted to the site [20].…”

“…Fusion of synaptojanin to a membrane targeting motif blocks epidermal-growth-factor (EGF)-receptor internalization [50], suggesting that constitutively active mutants of Rac may block endocytosis by recruiting synaptojanin to the plasma membrane and depleting PtdIns(4,5)P # at endocytic sites -but what does this say about how Rac acts in i o ? Rac is also able to stimulate synthesis of PtdIns(4,5)P # through activation of type I PtdIns4P 5-kinases [18,53]. The type I PtdIns4P 5-kinases provide PtdIns(4,5)P # for endocytosis [51] and the β-isoform is required for EGF-receptor internalization [54].…”

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).…”