Regulation of Ras Localization and Cell Transformation by Evolutionarily Conserved Palmitoyltransferases

Abstract

e Ras can act on the plasma membrane (PM) to mediate extracellular signaling and tumorigenesis. To identify key components controlling Ras PM localization, we performed an unbiased screen to seek Schizosaccharomyces pombe mutants with reduced PM Ras. Five mutants were found with mutations affecting the same gene, S. pombe erf2 (sp-erf2), encoding sp-Erf2, a palmitoyltransferase, with various activities. sp-Erf2 localizes to the trans-Golgi compartment, a process which is mediated by its third transmembrane domain and the Erf4 cofactor. In fission yeast, the human ortholog zDHHC9 rescues the phenotypes of sp-erf2 null cells. In contrast, expressing zDHHC14, another sp-Erf2-like human protein, did not rescue Ras1 mislocalization in these cells. Importantly, ZDHHC9 is widely overexpressed in cancers. Overexpressing ZDHHC9 promotes, while repressing it diminishes, Ras PM localization and transformation of mammalian cells. These data strongly demonstrate that sp-Erf2/zDHHC9 palmitoylates Ras proteins in a highly selective manner in the trans-Golgi compartment to facilitate PM targeting via the transGolgi network, a role that is most certainly critical for Ras-driven tumorigenesis. Mammals have three RAS genes, HRAS, NRAS, and KRAS, which encode membrane-bound GTPases that control a wide range of signal transduction pathways critical for growth, differentiation, and cytoskeleton remodeling (1). The best-known Ras function is to mediate growth factor signaling that occurs at the plasma membrane (PM), and deregulation of this and other Ras functions can promote tumor formation (2, 3). While all Ras proteins are farnesylated to gain general membrane affinity, a second signal is needed to specifically localize to the PM. For HRas and NRas, this signal is provided by palmitoylation at cysteine residues immediately upstream of the C-terminal CAAX motif. This reaction can be catalyzed by protein acyltransferases (PATs) that contain a DHHC domain rich with aspartate, histidine, and cysteine residues. These DHHC-PATs are expected to localize in the Golgi complex to allow the delivery of palmitoylated Ras proteins to the PM through the trans-Golgi system (4) although this concept has not been thoroughly demonstrated in vivo.Humans have 24 DHHC-PATs, but it remains unresolved which of these control palmitoylation of Ras proteins in vivo. In contrast, the Ras pathways are considerably simpler in yeasts. Early studies from the budding yeast Saccharomyces cerevisiae, which has seven DHHC-PATs and two Ras proteins, suggest that only S. cerevisiae Erf2 (sc-Erf2) controls palmitoylation of its Ras proteins (5, 6). By sequence alignment to find putative orthologs, zDHHC9 was predicted to be a human Ras PAT (7). Although zDHHC9 can catalyze Ras palmitoylation in vitro, zDHHC9 cannot rescue sc-erf2 mutant phenotypes in budding yeast (8), and until now there has been no evidence that zDHHC9 or other human DHHC-PATs can control Ras localization in mammalian cells. Furthermore, while ectopically expressed zDHHC9 localizes to the Golgi compl...

Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans

“…Additionally, S. cerevisiae Erf2 activity is dependent on Erf4 (3,31,33). However, our experiments suggest that CnPfa4, which is most similar to S. cerevisiae Pfa4, is the primary PAT for Ras1.…”

“…In addition, the localization of GFP-Ras1 at the PM was perturbed in the pfa4⌬ mutant but localized normally in the other PAT mutant strains. Although CnRas1 palmitoylation and PM localization were not completely eliminated in this singlemutant background, the observed level of reduction of Ras palmitoylation is similar to that in S. cerevisiae and S. pombe erf2 mutant strains (31,33).…”

“…cerevisiae) (31,33). To assess the localization of Pfa4, we generated a GFP-tagged version of this protein.…”

Mutations in the X-linked Intellectual Disability Gene, zDHHC9, Alter Autopalmitoylation Activity by Distinct Mechanisms

“…Indeed, reduction in the amount of zDHHC9 by expression of the microRNA (miR-134) in somatostatin interneurons affects the palmitoylation status and localization of Ras in these cells (31). In addition, palmitoyltransferase activity levels impart a basic mechanism for regulating substrate recognition and specificity in Schizosaccharomyces pombe (32) as well as regulating Ras plasma membrane localization (33). It appears that the amount and activity of DHHC palmitoyltransferases play a direct role in the levels and types of palmitoylated substrates.…”

Casein Kinase 1γ Ensures Monopolar Growth Polarity under Incomplete DNA Replication Downstream of Cds1 and Calcineurin in Fission Yeast

“…Like other posttranslational modifications, such as phosphorylation and acetylation, the palmitoylation reaction is also reversible (42), suggesting that this is another step by which Cki3 functions are regulated. Humans have 23 or 24 DHHC-containing proteins (42,43), and global proteomic analyses suggest that a plethora of proteins are palmitoylated in vivo (41,44,45). In budding and fission yeasts, the Erf2-Erf4 complex catalyzes modification of Ras and Rho family proteins (39,40,43,44,46,47).…”