Substrate recognition by the cell surface palmitoyl transferase DHHC5

Abstract

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.phospholemman | sodium pump | palmitoylation | DHHC | ion transport P rotein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol via a thioester bond, is catalyzed by Asp-His-His-Cys motif-containing palmitoyl acyltransferases (DHHC-PATs); there are 23 human isoforms (1). These zinc-finger-containing enzymes typically have four transmembrane (TM) domains, with a conserved ∼50 amino acid cysteine-rich cytosolic core located between TM2 and -3, which contains a conserved DHHC motif, the active site. In contrast, the intracellular amino and carboxyl termini are poorly conserved, and likely contribute to DHHC isoform substrate selectivity (1). DHHC-PATs are expressed throughout the secretory pathway, but DHHC5 is widely recognized as one of very few cell-surfacelocalized PATs (2, 3). The final four amino acids of DHHC5 form a canonical class II PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, which interacts with postsynaptic density protein 95 (PSD-95) (2), although PSD-95 is not itself a DHHC5 substrate.An appreciation is now growing that protein palmitoylation turns over rapidly (in minutes) for certain proteins (4-8). For example, dynamic surface membrane protein palmitoylation by DHHC5 underlies a novel form of endocytosis, massive endocytosis (MEND), in which up to 70% of the cell surface membrane is internalized (7, 8). Calci...

Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation

“…S3A). With respect to the PEG-switch assay (PSA) (18), the reported conditions showed incomplete NH 2 OHdependent PEGylation of CANX, Ras, and IFITM3 (lanes 2 and 8, Fig. S3B).…”

Understanding the rules governing NCX1 palmitoylation

“…8 This highlights the difficulties of predicting palmitoylation sites in proteins: the recognition rules that apply to one DHHC-PAT may apply to closely related family members, but not to all enzymes. From a therapeutic point of view this is good news, because it means we should be able to specifically target individual DHHC-PATs, or even individual substrates, but there is still a long way to go before palmitoylation sites can be accurately predicted.…”

All about that fat: Lipid modification of proteins in Cryptococcus neoformans

“…Possible explanations for this widespread apparent redundancy include differing subcellular localization of the enzymes that enables spatial control of protein modification (Ohno et al , 2006; Howie et al , 2014), or distinct substrate specificity (Roth et al , 2006; Ohno et al , 2012). Although the mechanism of specificity remains elusive, recent work in several pathogens, including C. neoformans , has identified specific PATs that regulate critical pathogenic processes through the modification of unique protein substrates (Jones et al , 2012; Frenal et al , 2013; Santiago-Tirado et al , 2015).…”