Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

Akiyuki Nishimura,John J.G Tesmer,Mohamed Aittaleb,Maurine E Linder

doi:10.1074/jbc.m111.273342

Akiyuki Nishimura, John J.G Tesmer + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.273342

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Abstract

Activation of G protein-coupled receptors at the cell surface leads to the activation or inhibition of intracellular effector enzymes, which include various Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs activate small molecular weight GTPases at the plasma membrane (PM). Many of the known G protein-coupled receptor-regulated RhoGEFs are found in the cytoplasm of unstimulated cells, and PM recruitment is a critical aspect of their regulation. In contrast, p63RhoGEF, a Gα(q)-regulated RhoGEF, appears to be constitutively localized to the PM. The objective of this study was to determine the molecular basis for the localization of p63RhoGEF and the impact of its subcellular localization on its regulation by Gα(q). Herein, we show that the pleckstrin homology domain of p63RhoGEF is not involved in its PM targeting. Instead, a conserved string of cysteines (Cys-23/25/26) at the N terminus of the enzyme is palmitoylated and required for membrane localization and full basal activity in cells. Conversion of these residues to serine relocates p63RhoGEF from the PM to the cytoplasm, diminishes its basal activity, and eliminates palmitoylation. The activity of palmitoylation-deficient p63RhoGEF can be rescued by targeting to the PM by fusion with tandem phospholipase C-δ1 pleckstrin homology domains or by co-expression with wild-type Gα(q) but not with palmitoylation-deficient Gα(q). Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by Gα(q), as opposed to other known Gα-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their high basal activity.

Highlights

Rho GTPases act as molecular switches that mediate a wide variety of cellular processes, such as actin cytoskeletal organization, cell migration, cell cycle progression, and transcriptional control [1,2,3]
To identify the region responsible for targeting p63RhoGEF to the plasma membrane (PM) (Fig. 2A), we designed a series of truncation and deletion mutants with an N-terminal EGFP fusion (Fig. 1) and examined their subcellular localization by confocal microscopy
pleckstrin homology (PH) domains are widely recognized for their ability to interact with phospholipid headgroups [30, 31], deletion of the PH domain did not disrupt PM localization of p63RhoGEF (Fig. 2B)

Summary

Introduction

Rho GTPases act as molecular switches that mediate a wide variety of cellular processes, such as actin cytoskeletal organization, cell migration, cell cycle progression, and transcriptional control [1,2,3]. RH-RhoGEFs are distributed throughout the cytoplasm of unstimulated cells and translocate to the PM upon activation of G␣12 or G␣13 subunits (19 –21) Direct activation of these RhoGEFs has been difficult to demonstrate in vitro with the exception of p115RhoGEF [22], suggesting that the dominant mechanism by which G␣12/13 subunits activate RHRhoGEFs is via membrane recruitment. This would be consistent with their high basal activity [23] and the fact that G␣12/13 subunits bind primarily to the RH domain, which is located ϳ200 amino acids N-terminal to the catalytic DH/PH tandem domains [24]. Because wild-type G␣q can achieve levels of activation comparable with deletion of the p63RhoGEF PH domain, even in the case of palmϪ p63RhoGEF, the chief mechanism regulating p63RhoGEF activity seems to be allosteric and probably does not involve localization to specific locales on the membrane surface or reorientation of p63RhoGEF for more optimal interactions with RhoA

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