Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex

Significance The stimulation of certain surface receptors on immune cells triggers the release of calcium (Ca ²⁺) stored in the endoplasmic reticulum (ER). This Ca ²⁺ flux is required for efficient activation and function of immune cells, and involves the ER membrane Ca ²⁺ channel, the inositol 1,4,5-triphosphate receptor (IP3R). We found that stable expression of IP3R requires the addition of a fatty acid through a process called palmitoylation catalyzed by an enzyme complex composed of DHHC6 (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) and selenoprotein K (Selk) proteins. These findings provide new mechanistic insight into the selenium-sensitive fine-tuning of immune cell activation through posttranslational modification of the IP3R Ca ²⁺ channel. This study also reveals a novel DHHC6/Selk enzyme complex responsible for regulating stable expression of the IP3R. Calcium (Ca ²⁺) is a secondary messenger in cells and Ca ²⁺ flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca ²⁺ flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca ²⁺ flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially p