Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex Gregory J. Fredericksa, FuKun W. Hoffmanna, Aaron H. Rosea, Hanna J. Osterheldb, Franz M. Hessc, Frederic Mercierd, and Peter R. Hoffmanna,1 Departments of aCell and Molecular Biology, cMedicine, and dTropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813; and bBiotechnology Department, University of Applied Sciences Mannheim, 68163 Mannheim, Germany Edited by Anjana Rao, Sanford Consortium for Regenerative Medicine and La Jolla Institute for Allergy and Immunology, La Jolla, CA, and approved October 13, 2014 (received for review September 5, 2014) + + + Calcium (Ca2 ) is a secondary messenger in cells and Ca2 flux ini- release-activated Ca2 (CRAC) channels. These steps are collec- + tiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-tri- tively referred to as store-operated Ca2 entry (SOCE) and defects phosphate (IP3) binding to the IP3 receptor (IP3R) is particularly in any of the factors involved in SOCE significantly impair immune important for the activation and function of immune cells. Previous cell function (8). studies demonstrated that genetic deletion of selenoprotein K The IP3R family includes three isoforms with multiple splice (Selk) led to decreased Ca2+ flux in a variety of immune cells and variants, and all IP3R isoforms share a tetrameric architecture impaired immunity, but the mechanism was unclear. Here we show consisting of an N-terminal ligand binding domain and a C-ter- that Selk deficiency does not affect receptor-induced IP3 produc- minal channel domain containing six transmembrane domains tion, but Selk deficiency through genetic deletion or low selenium (9, 10). The intervening region is referred to as the regulatory or in culture media leads to low expression of the IP3R due to a defect coupling domain and structure/function studies have revealed in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters important insights into how IP3 binding at the N-terminal region represent the amino acids aspartic acid, histidine, histidine, and leads to channel opening in the C-terminal region (11). There cysteine in the catalytic domain) family of enzymes that catalyze is evidence to suggest that the IP3Rs are arranged in the ER protein palmitoylation revealed that one member, DHHC6, contains membrane in a nonrandom distribution and that dynamic clus- a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to tering of these receptors may be functionally important (12, 13). Phosphorylation and dephosphorylation of the IP3R also regulates the ER membrane. Because Selk is also an ER membrane protein 2+ and contains an SH3 binding domain, immunofluorescence and Ca flux through this channel (14), but it remains to be de- coimmunoprecipitation experiments were conducted and revealed termined whether the IP3R is regulated by other posttranslational modifications. Given the central role that the IP3R plays in SOCE DHHC6/Selk interactions in the ER membrane that depended on 2+ SH3/SH3 binding domain interactions. DHHC6 knockdown using in so many immune cell types, expression of this Ca channel shRNA in stably transfected cell lines led to decreased expression of protein represents a crucial point of regulating immune responses. 2+ Our previous studies have demonstrated that low levels of the IP3R and impaired IP3R-dependent Ca flux. Mass spectrophoto- dietary selenium as well as a deficiency in a specific ER-localized metric and bioinformatic analyses of the IP3R protein identified two selenium-containing protein, selenoprotein K (Selk), led to im- palmitoylated cysteine residues and another potentially palmitoy- paired SOCE (15, 16). The specific mechanism by which Selk + lated cysteine, and mutation of these three cysteines to alanines deficiency affected Ca2 flux was not apparent, but the fact that resulted in decreased IP3R palmitoylation and function. These findings + reveal IP3R palmitoylation as a critical regulator of Ca2 flux in im- mune cells and define a previously unidentified DHHC/Selk com- Significance plex responsible for this process. The stimulation of certain surface receptors on immune cells 2+ selenium | calcium | immune | endoplasmic reticulum | palmitoylation triggers the release of calcium (Ca ) stored in the endoplasmic reticulum (ER). This Ca2+ flux is required for efficient activation and function of immune cells, and involves the ER membrane mmune cell activation relies on receptor-mediated increases in + + Ca2 channel, the inositol 1,4,5-triphosphate receptor (IP3R). We cellular calcium (Ca2 ) concentrations, which is an indispens- I found that stable expression of IP3R requires the addition of able step in proliferation, differentiation, migration, and effector + a fatty acid through a process called palmitoylation catalyzed by functions during immune responses (1, 2). A rapid influx of Ca2 an enzyme complex composed of DHHC6 (letters represent the has been shown to be important during the activation of lympho- amino acids aspartic acid, histidine, histidine, and cysteine in the cytes through the T- and B-cell receptors, macrophages through catalytic domain) and selenoprotein K (Selk) proteins. These Fcγ receptors, and mast cells through Fce receptors and stimula- findings provide new mechanistic insight into the selenium- tion of various immune cells through chemokine receptors (3). sensitive fine-tuning of immune cell activation through post- Engagement of these receptors at the plasma membrane leads to 2+ translational modification of the IP3R Ca channel. This study the activation of phosphoinositide-specific phospholipase C, which also reveals a novel DHHC6/Selk enzyme complex responsible catalyzes the degradation of phosphatidylinositol-4,5-bisphosphate for regulating stable expression of the IP3R. to generate inositol 1,4,5-triphosphate (IP3) and diacylglycerol (4). IP3 binds to the IP3 receptor (IP3R) in the endoplasmic reticulum Author contributions: G.J.F., F.W.H., and P.R.H. designed research; G.J.F., F.W.H., A.H.R., 2+ (ER) membrane leading to Ca mobilization from the ER, which H.J.O., F.M.H., F.M., and P.R.H. performed research; G.J.F., F.W.H., A.H.R., and P.R.H. + + is the main Ca2 store in immune cells (5). The release of Ca2 analyzed data; and P.R.H. wrote the paper. from the ER lumen to the cytosol causes structural changes and The authors declare no conflict of interest. oligomerization of the ER transmembrane protein, stromal in- This article is a PNAS Direct Submission. teraction molecule 1 (STIM1) (6, 7). These changes allow the cy- 1To whom correspondence should be addressed. Email: [email protected]. tosolic domain of STIM1 to directly interact with the pore-forming This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ 2+ unit, Orai1, of Ca channels on the plasma membrane called Ca 1073/pnas.1417176111/-/DCSupplemental. 16478–16483 | PNAS | November 18, 2014 | vol. 111 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1417176111 Downloaded by guest on September 27, 2021 2+ Ca induced with thapsigargin was not altered in Selk-deficient Macrophages T cells A WT immune cells suggested Selk may affect SOCE through the 0.2 WT 0.08 WT 0.8 Selk KO Selk KO Selk KO actions of IP3R. Subsequently, we found that Selk expression M) 0.6 µ M) was required for the stable expression of a different membrane µ 0.1 0.04 0.4 IP3 ( receptor, the low-density lipoprotein receptor CD36 (17). This IP3 ( 0.2 study revealed that Selk was required for palmitoylation of CD36, 0 0 0 and Selk-deficient macrophages exhibited lower levels of CD36 Stimulation: None anti- due to impaired palmitoylation. CD3/CD28 Palmitoylation is a posttranslational modification involving the Macrophages T cells B 1.20 reversible addition of the 16-carbon fatty acid, palmitate, to 1.20 WT 1.401.4 WT uncaged IP3 KO uncaged IP3 KO 1.151.15 cysteine residues through a thioester bond (18), and this modi- 1.301.3 1.101.10 fication can facilitate membrane association of proteins or stable 1.201.2 expression of transmembrane proteins (19, 20). The requirement 1.051.05 Flux (F1/F0) 2+ Flux (F1/F0) 1.101.1 2+ of Selk for palmitoylation of CD36 and the impaired Ca flux in 1.001.00 2+ Ca Ca 1.0 Selk-deficient immune cells led us to explore the possibility that 0.950.95 1.00 the IP3R is palmitoylated in a Selk-dependent manner and that 0.90 0.9 0.90 0 10 20 30 40 50 0.90 01020 30 40 50 0200 20 4040 6060 8080 100100 110120 120140 this is required for its stable expression. The data presented in Time (s) Time (s) the current study reveal that IP3R does indeed require palmi- toylation for stable expression, and our data identify DHHC6 Fig. 1. Selk deficiency does not affect IP3 production but decreases IP3R (letters represent the amino acids aspartic acid, histidine, histi- function. (A) BMDMs or T cells from WT or Selk KO mice were analyzed for dine, and cysteine in the catalytic domain) as a palmitoyl acyl levels of IP3 after stimulation with either 100 ng/mL MCP-1 or immune com- transferase enzyme that interacts with Selk to carry out the pal- plexes (BMDM, Left) or plate-bound CD3/CD28 (T cells, Right). IP3 after stim- mitoylation of specific cysteine residues within the IP3R in immune ulation was similar between WT and Selk KO for both BMDMs and T cells. (B) cells. These findings provide previously unidentified mechanistic BMDM (Left)orTcells(Right)fromWTorSelkKOmicewereloadedwith insight into the fine tuning of immune cell activation and immune fluo4-acetoxymethyl ester (fluo4-AM) and caged IP3 and change in fluores- 2+ cence was measured after flash UV-induced uncaging of the IP3. For both responses through posttranslational modification of the IP3R Ca + BMDMs and T cells, Selk KO cells exhibited decreased Ca2 flux compared with channel and uncover a novel enzyme complex responsible for + regulating stable expression of the IP3R.