Identification of a Novel Prenyl and Palmitoyl Modification at the CaaX Motif of Cdc42 That Regulates RhoGDI Binding

Abstract

Membrane localization of Rho GTPases is essential for their biological functions and is dictated in part by a series of posttranslational modifications at a carboxyl-terminal CaaX motif: prenylation at cysteine, proteolysis of the aaX tripeptide, and carboxymethylation. The fidelity and variability of these CaaX processing steps are uncertain. The brain-specific splice variant of Cdc42 (bCdc42) terminates in a CCIF sequence. Here we show that brain Cdc42 undergoes two different types of posttranslational modification: classical CaaX processing or novel tandem prenylation and palmitoylation at the CCaX cysteines. In the dual lipidation pathway, bCdc42 was prenylated, but it bypassed proteolysis and carboxymethylation to undergo modification with palmitate at the second cysteine. The alternative postprenylation processing fates were conserved in the GTPases RalA and RalB and the phosphatase PRL-3, proteins terminating in a CCaX motif. The differentially modified forms of bCdc42 displayed functional differences. Prenylated and palmitoylated brain Cdc42 did not interact with RhoGDI␣ and was enriched in the plasma membrane relative to the classically processed form. The alternative processing of prenylated CCaX motif proteins by palmitoylation or by endoproteolysis and methylation expands the diversity of signaling GTPases and enables another level of regulation through reversible modification with palmitate.

Regulating Rho GTPases and their regulators

“…Palmitoylation has also been described for the brain-specific splice variant of CDC42 (bCDC42) 20 . CDC42 has many cellular functions, including regulation of cell polarity, extension of filopodia and vesicle trafficking.…”

“…The bCDC42 isoform terminates with a CCIF sequence and, after prenylation at Cys188, bypasses C-terminal proteolysis and carboxymethylation and undergoes palmitoylation at the adjacent Cys189 residue 20 . Interestingly, this dual-lipidated bCDC42 does not interact with RhoGDI and is therefore enriched at the plasma membrane, compared to a palmitoylation-deficient form 20 .…”

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

“…Dual Lipid Modification of the Ral CAAX Motif Affects RalB Subcellular Localization-In addition to the conventional modification pathway involving RCE1 and ICMT, RalA and RalB can also undergo an alternative pathway, whereby the newly GGTase I-modified protein does not undergo subsequent RCE1-mediated proteolytic removal of the A 1 A 2 X residues but instead undergoes palmitoylation of the cysteine residue at the A 1 position (26). Although the mechanism that regulates which pathway will be taken has not been elucidated, it was reported that the level of palmitoylation of RalA was increased in RCE1-deficient MEFs.…”

“…lized by Nishimura and Linder (26) in defining the pathway. To do so, we introduced a Cys to Ser amino acid substitution at the A 1 position of each Ral isoform (C204S), designated RalA-CSIL and RalB-CSLL.…”

“…A recent study of the brain-specific isoform of the Rho family small GTPase Cdc42 concluded that proteins terminating in a CA 1 A 2 X motif in which the A 1 residue is a cysteine (CA 1 A 2 X ϭ CCAX) can undergo an alternative processing pathway (26). Although a majority of Cdc42 undergoes the conventional post-prenyl modification pathway involving RCE1 and ICMT, a subset (5-20%) does not undergo RCE1-catalyzed removal of the AAX residues, and instead the A 1 cysteine is covalently modified by palmitoylation.…”