Dual Regulation of RA-RhoGAP Activity by Phosphatidic Acid and Rap1 during Neurite Outgrowth

Abstract

During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth.Formation and extension of axons and dendrites, the socalled neurite outgrowth, are crucial events in neuronal differentiation and maturation during development of the nervous system (1, 2). As neurites extend further and acquire their final axonal and dendritic identities, neurons establish synaptic contacts and reach full maturation (3-5). These morphological changes require remodeling of the actin cytoskeleton (6). Rho small G protein is a key regulator of the actin cytoskeleton organization in neurons and has been shown to play important roles in several aspects of neurite outgrowth, such as sprouting, extension, and branching (7,8). An increase in Rho activity results in reduction of neurite sprouting, extension, and branching, whereas a decrease in Rho activity enhances neurite sprouting, extension, and branching (9 -12). The Rho activity is positively regulated by guanine nucleotide exchange factors (GEFs) 2 and negatively regulated

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…As with several other PtdOH-protein interactions, mutation of a basic residue, arginine 399, abolished PtdOH binding. The interaction of RA-RhoGAP with PtdOH-stimulated GAP activity in vitro (Kurooka et al, 2011). Overexpression of DAG kinase increased neurite length in a manner that depended on PtdOH binding to RA-RhoGAP.…”

“…As key regulators of cytoskeleton dynamics, Rho GTPases are known to control neurite development and decreased Rho activity is associated with enhanced neurite sprouting, extension, and branching (Luo, 2000). RARhoGAP is a Rho GAP that binds PtdOH via its PH domain (Kurooka et al, 2011). As with several other PtdOH-protein interactions, mutation of a basic residue, arginine 399, abolished PtdOH binding.…”

A GEF-to-phospholipase molecular switch caused by PA, RAC and jak tyrosine kinase, that explains leukocyte cell migration

“…Liposome co-sedimentation assay was performed as explained in (Kurooka et al, 2011;Lee et al, 2002). Liposomes composed of phosphatidylcholine (PC), both PC and PA, both PC and PI(3)P or both PC and PI(4,5)P2 (weight ratio 6:4) were resuspended in 100 ml buffer (50 mM Tris-HCl, pH 7.4 and 100 mM NaCl) to which 1 mg of myc-PLD2WT or GST-PX or GST-PH was added.…”

Regulating G protein activity by lipase-independent functions of phospholipase C

“…PA was found to enhance the stimulation of ASAP1 GAP by PIP 2 , markedly increasing Arf GTPase activity in vitro [53]. PA also increased the activity of Rasassociating domain-RhoA GAP (RA-RhoA GAP), alone and through synergism with Rap1, dependent on binding to the PH domain [59].…”