Article

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

Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2011; 286(39):34448-56. DOI: 10.1074/jbc.M111.273342
Source: PubMed

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 Gαq-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 Gαq. 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
Gαq but not with palmitoylation-deficient Gαq. Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by Gαq, as opposed to other known Gα-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their
high basal activity.

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Available from: Mohamed Aittaleb, May 06, 2015
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    • "To address the localization and dynamics of p63RhoGEF and its splice variant GEFT in living cells, fusions with fluorescent proteins were made (Figure 1). The full-length p63RhoGEF fused to the YFP variant mVenus1617, denoted as YFP-p63, displayed clear plasma membrane localization in living HeLa cells as can be inferred from confocal images depicted in Figure 2A and as was observed previously15. Plasma membrane localization was observed when YFP was fused to the C-terminus of p63RhoGEF (p63-YFP) (Figure 2A), indicating that plasma membrane labeling was independent of the location of the fluorescent protein. In contrast, the splice variant, GEFT, did not localize at the membrane but was located in the cytoplasm and was largely excluded from the nucleus (Figure 2A). "
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