NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.

Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT

Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in Pdx1-Cre;LSL-KrasG12D mice, suggesting that ICMT is required for NOTCH signaling. We used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies). We identified Rab7 and Rab8 as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.

Alterations in molecular chaperones and eIF2α during lung endothelial cell apoptosis

We have previously demonstrated that inhibition of CAAX carboxyl methylation with AGGC caused redistribution and condensation of the ER molecular chaperones, glucose-regulated protein (GRP)-94 and calnexin; an effect that was attenuated by overexpression of dominant active RhoA. We have also shown that AGGC decreased GRP94 protein level; an effect that was dependent on caspase activity. In the present study, we tested the effects of inhibition of posttranslational processing of CAAX proteins on localization and protein levels of molecular chaperones and phosphorylation and protein level of eIF2α. We found that both AGGC, which inhibits CAAX carboxyl methylation, and simvastatin, which inhibits CAAX geranylgeranylation, caused relocalization of GRP94, calnexin, and calreticulin, effects that were not seen during endothelial apoptosis induced by TNF-α or ultraviolet (UV) irradiation. These results suggest that posttranslational processing of CAAX proteins is important in maintaining localization of molecular chaperones normally found in the ER. We also noted that AGGC, but not simvastatin, TNF-α, or UV irradiation, decreased protein levels of most molecular chaperones. Increased eIF2α phosphorylation was observed in the early stages of apoptosis, which was independent of the cause of apoptosis. These results suggest that eIF2α phosphorylation is a common early response to apoptosis-inducing stimuli. Interestingly, eIF2α protein level was decreased in the late stages of apoptosis induced by AGGC, TNF-α, and UV irradiation: an effect that was prevented by caspase inhibition. Thus we speculate that caspase(s)-dependent proteolysis of molecular chaperones and eIF2α may be novel signaling pathways of apoptosis. We also speculate that increased eIF2α phosphorylation is a defensive response against endothelial cell apoptosis.

Protein carboxyl methylation and the biochemistry of memory

Bacterial chemotaxis is mediated by two reversible protein modification chemistries: phosphorylation and carboxyl methylation. Attractants bind to membrane chemoreceptors that control the activity of a protein kinase which acts in turn to control flagellar motor activity. Coordinate changes in receptor carboxyl methylation provide a negative feedback mechanism that serves a memory function. Protein carboxyl methylation might play an analogous role in the nervous system. Two protein carboxyl methyltransferases serve to regulate signal transduction pathways in eukaryotic cells. One is highly expressed in the Purkinje layer of the cerebellum where it methyl esterifies prenylated cysteine residues at the carboxyl-termini of Ras-related and heterotrimeric G-proteins. The other is abundant throughout the brain where it methylates the carboxyl-terminus of protein phosphatase 2A. The phosphatase methyltransferase and the protein methylesterase that reverses phosphatase methylation are structurally related to the corresponding bacterial chemotaxis methylating and demethylating enzymes. Recent results indicate that deficiencies in phosphatase methylation play an important role in the etiology of Alzheimer's disease.

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets

ABSTRACT The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either T. brucei CaaX protease when examined in the context of the yeast a-factor reporter but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that T. brucei Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases and that a subset of these compounds disrupt T. brucei Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both T. brucei CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences T. brucei CaaX protease specificity.