Signal transduction down the Ras/MAPK pathway, including that critical to T cell activation, proliferation, and differentiation, has been generally considered to occur at the plasma membrane. It is now clear that the plasma membrane does not represent the only platform for Ras/MAPK signaling. Moreover, the plasma membrane itself is no longer considered a uniform structure but rather a patchwork of microdomains that can compartmentalize signaling. Signaling on internal membranes was first recognized on endosomes. Genetically encoded fluorescent probes for signaling events such as GTP/GDP exchange on Ras have revealed signaling on a variety of intracellular membranes, including the Golgi apparatus. In fibroblasts, Ras is activated on the plasma membrane and Golgi with distinct kinetics. The pathway by which Golgi-associated Ras becomes activated involves PLCγ and RasGRP1 and may also require retrograde trafficking of Ras from the plasma membrane to the Golgi as a consequence of depalmitoylation. Thu...

Compartmentalized Ras/MAPK signaling.

PROTEINS INVOLVED IN PRENYL-DEPENDENT PROCESSING 217 Protein Prenyltransferases 218 Proteases 221 Reversible Modifications 223 PRENYL-MEDIATED INTERACTIONS 225 Prenyl-protein Receptors 225 Lipid Interactions 227 Indirect Prenyl-dependent Targeting 229

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Protein prenylation: genes, enzymes, targets, and functions.

Prenylated proteins contain a covalently linked cholesterol intermediate near their carboxyl-termini. Maturation of most prenylated proteins involves proteolytic removal of the last three amino acids. Two genes in Saccharomyces cerevisiae, RCE1 and AFC1, were identified that appear to be responsible for this processing. The Afc1 protein is a zinc protease that participates in the processing of yeast a-factor mating pheromone. The Rce1 protein contributes to the processing of both Ras protein and a-factor. Deletion of both AFC1 and RCE1 resulted in the loss of proteolytic processing of prenylated proteins. Disruption of RCE1 led to defects in Ras localization and signaling and suppressed the activated phenotype associated with the allele RAS2val19.

https://science.sciencemag.org/content/sci/275/5307/1796.full.pdf

Modulation of Ras and a-factor function by carboxyl-terminal proteolysis

RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylation-processing steps--particularly that of ICMT-catalysed methylation--might provide a better approach to the control of cancer-cell proliferation.

Post-prenylation-processing enzymes as new targets in oncogenesis

INTRODUCTION . . . . . ... . .... . . . . . . . . . . . . . . . ... . ..... . . . . ... 1 8 1 MODIFICATIONS O F THE N O R C -TERMINI O F RAS-RELATED GTPASES . . . . 182 FUNCTIONAL SIGNIFICANCE OF THE MODIFICATIONS . . . . . . . . . . . . . . . . . . . . 187 ARF Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 87 Ras Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 Rap Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Rho-related Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 Rab Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 CONCLUSIONSIFUTURE DIRECTIONS 197

Role of Protein Modification Reactions in Programming Interactions Between Ras-Related GTPases and Cell Membranes

Retinal transducin was previously shown to be farnesylated on its gamma subunit. This farnesylation reaction on a cysteine residue near the carboxyl terminus is followed by peptidase cleavage at the cysteine. Thus the modified cysteine becomes the carboxyl terminus. It is shown here that the free carboxyl group can be methylated by an S-adenosyl-L-methionine-dependent methyltransferase associated with the rod outer segment membranes. This process can be inhibited by S-adenosyl-L-homocysteine and sinefungin. Moreover, synthetic N-acetyl-S-farnesyl-L-cysteine, but not N-acetyl-L-cysteine, is a substrate for the enzyme. Rapid demethylation of N-acetyl-S-farnesyl-L-cysteine methyl ester can be observed in the membranes. Transducin is also enzymatically demethylated by the rod outer segment membranes. Moreover, the 23- to 29-kDa small G proteins are methylated and demethylated in this system. These data suggest that methylation/demethylation may play a regulatory role in visual signal transduction.

https://www.pnas.org/content/pnas/88/8/3043.full.pdf

Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments.

Identifying the recognition unit for G protein methylation.

Proteins from eucaryotic cells which have a carboxyl-terminal CAAX motif are posttranslationally modified by isoprenylation. The pathway involves the linkage of an all-trans-farnesyl (C15) or an all-trans-geranylgeranyl (C20) moiety to the cysteine residue followed by proteolysis which generates the modified cysteine as the carboxyl-terminal residue. Carboxylmethylation of the modified cysteine residue completes the pathway. This latter methylation reaction is the only potentially reversible reaction in the pathway and thus of possible regulatory significance. A specific esterase is required to reverse the methylation. It is demonstrated here that simple isoprenylated cysteine derivatives, such as N-acetyl-S-farnesyl-L-cysteine methyl ester (L-AFCM) and N-acetyl-S-geranylgeranyl-L-cysteine methyl ester (L-AGGCM), are substrates for a rod outer segment (ROS) membrane esterase activity. The KM and Vmax values for L-AFCM and L-AGGCM are 186 microM and 2.2 nmol mg-1 min-1 and 435 microM and 4.8 nmol mg-1 min-1, respectively. The enzyme(s) is stereoselective rather than stereospecific because D-AFCM is enzymatically hydrolyzed with KM and Vmax values of 157 microM and 0.46 nmol mg-1 min-1, respectively. The enzyme(s) does not process N-acetyl-L-cysteine methyl ester, demonstrating that the isoprenyl moiety is required for substrate activity. Ebelactone B is a potent mechanism-based inactivator of the enzyme with a KI = 42 microM and a kinh = 3.7 x 10(-3) s-1. Importantly, L-AFCM, L-AGGCM, and ebelactone B all inhibit the demethylation of the endogenous ROS substrates, showing that the same enzymatic activity is involved in the processing of the synthetic and physiological substrates.

Identification of an isoprenylated cysteine methyl ester hydrolase activity in bovine rod outer segment membranes.

Proteins that are post-translationally modified by prenylation can be either farnesylated (C-15) or geranylgeranylated (C-20) by separate prenyltransferase enzymes. Prenylated proteins are also methylated at their C-terminal residue by S-adenosylmethionine-linked methylation. In this paper we show that the methylation of farnesylated and geranyl-geranylated substrates can be accounted for by the presence of a single enzyme. It is demonstrated that the Km and Vmax. values for the retinal rod outer segment methyltransferase, measured with small molecule farnesylated and geranylgeranylated substrates, are identical. These substrates mutually inhibit each other's methylation, with KI values being equal to their Km values. The Km for S-adenosylmethionine was measured to be the same with either farnesylated or geranylgeranylated substrates. Competitive inhibitors of the methyltransferase containing either a geranylgeranyl or a farnesyl group equally block the methylation of synthetic geranylgeranylated and farnesylated substrates of the enzyme. Importantly, these inhibitors are also equipotent at inhibiting the methylation of the physiological substrates of the rod outer segment methyltransferase. These substrates are both farnesylated and geranylgeranylated. One of these substrates had previously been identified as the farnesylated gamma subunit of transducin. Therefore it appears that the same enzymic activity can methylate both farnesylated and geranylgeranylated substrates.

Eng Wui Tan

Papers

Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments.

Identifying the recognition unit for G protein methylation.

Identification of an isoprenylated cysteine methyl ester hydrolase activity in bovine rod outer segment membranes.

Prenylated protein methyltransferases do not distinguish between farnesylated and geranylgeranylated substrates