Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates + Ca2 dynamics in T cells Jin Seok Wooa,1, Sonal Srikantha,1, Miyuki Nishib, Peipei Pinga, Hiroshi Takeshimab, and Yousang Gwacka,2 aDepartment of Physiology, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA 90095; and bGraduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan Edited by Arthur Weiss, University of California, San Francisco, CA, and approved February 2, 2016 (received for review December 9, 2015) Orai1 and stromal interaction molecule 1 (STIM1) mediate store-oper- E-Syt proteins were shown to be important for PIP2 replenishment ated Ca2+ entry (SOCE) in immune cells. STIM1, an endoplasmic re- after store depletion (17). The importance of protein interaction in ticulum (ER) Ca2+ sensor, detects store depletion and interacts with STIM1 recruitment was demonstrated by a STIM1ΔKmutant plasma membrane (PM)-resident Orai1 channels at the ER–PM junc- truncated in its C-terminal polybasic domain. Interaction with tions. However, the molecular composition of these junctions in T cells Orai1 or junctate facilitated recruitment of this PIP2 binding- remains poorly understood. Here, we show that junctophilin-4 (JP4), a deficient mutant into the junctions (15, 18, 19). It was thought that member of junctional proteins in excitable cells, is expressed in T cells the roles of dyad/triad junctional proteins are limited to muscle + and localized at the ER–PM junctions to regulate Ca2 signaling. Silenc- cells. However, identification of junctate as a STIM1-interacting + – ing or genetic manipulation of JP4 decreased ER Ca2 content and partner implied that some components (or homologs) of ER PM SOCE in T cells, impaired activation of the nuclear factor of activated junctions in excitable cells may be shared in immune cells. T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling The junctophilin family consists of four genes (JP1, JP2, JP3, and JP4) that are expressed in a tissue-specific manner and are pathways, and diminished expression of activation markers and cyto- – kines. Mechanistically, JP4 directly interacted with STIM1 via its cyto- known to form ER PM junctions in excitable cells (20, 21). plasmic domain and facilitated its recruitment into the junctions. Junctophilins contain eight repeats of the membrane occupation Accordingly, expression of this cytoplasmic fragment of JP4 inhibited and recognition nexus (MORN) motifs that bind to phospho- lipids in the N terminus and a C-terminal ER membrane-span- SOCE. Furthermore, JP4 also formed a complex with junctate, a + ning transmembrane segment (20, 22). In this study, we observed Ca2 -sensing ER-resident protein, previously shown to mediate STIM1 expression of JP4 in both human and mouse T cells, which was recruitment into the junctions. We propose that the junctate–JP4 com- further enhanced by TCR stimulation. Depletion or deficiency plex located at the junctions cooperatively interacts with STIM1 to 2+ 2+ of JP4 reduced ER Ca content, SOCE, and activation of the maintain ER Ca homeostasis and mediate SOCE in T cells. nuclear factor of activated T cells (NFAT) and ERK mitogen- activated protein kinase (MAPK) pathways. Mechanistically, JP4 – 2+ junctophilins | ER PM junctions | store-operated Ca entry | STIM1 | Orai1 depletion reduced accumulation of STIM1 at the junctions without affecting the number and length of the ER–PM junctions. We ob- he endoplasmic reticulum (ER)–plasma membrane (PM) served a direct interaction between the cytoplasmic regions of JP4 Tjunctions are ubiquitous structures essential for intermembrane and STIM1, and, correspondingly, overexpression of the STIM1- communications (1–3). These junctions play an important role in interacting JP4 fragment had a dominant negative effect on SOCE. + + lipid transfer and regulation of Ca2 dynamics, including ER Ca2 Finally, we identified a protein complex consisting of JP4 and + homeostasis and Ca2 entry after receptor stimulation (1, 4). junctate at the ER–PM junctions, which may have a synergistic Four major categories of components of the ER–PM junctions have been identified so far: (i) dyad/triad junctional proteins in Significance the heart and skeletal muscle (e.g., junctophilins and junctin), (ii) ER-resident vesicle-associated membrane protein-associated pro- Distinct membranes separate cellular organelles, and communication teins (VAPs) that form the lipid transfer machinery by interacting between organelles occurs primarily at the interorganelle mem- with phospholipid-binding proteins, (iii) extended synaptogamin- brane junctions, which are established by junctional proteins. The like proteins (E-Syts) that tether membranes, and (iv)theOrai1– junctions between the endoplasmic reticulum (ER) and the plasma + stromal interaction molecule 1 (STIM1) complex that forms the membrane (PM) are essential for lipid transfer and Ca2 dynamics; + + + primary Ca2 channel in T cells, the Ca2 release-activated Ca2 however, little is known about the composition of these junctions in (CRAC) channels. Among these proteins, the dyad/triad junctional T cells. We identified a protein complex containing junctophilin-4 proteins and the Orai1–STIM1 complex are known to play a crucial and junctate as components of the ER–PM junctions that regulate + + role in Ca2 dynamics, including excitation–contraction coupling Ca2 dynamics in T cells by interacting with stromal interaction + + in muscle and store-operated Ca2 entry (SOCE) in immune molecule 1 (STIM1), an essential activator of store-operated Ca2 cells, respectively (2, 5). channels. This study highlights an important role of junctional pro- Stimulation of T-cell receptors (TCRs) triggers activation of teins in T cells and helps in uncovering the pathological mechanisms SOCE primarily mediated by the PM-resident Orai1 channels underlying human diseases due to mutations in these proteins. + and ER-resident STIM1 protein that senses ER Ca2 concen- tration (6–11). Upon store depletion, STIM1 translocates and Author contributions: J.S.W., S.S., and Y.G. designed research; J.S.W. and S.S. performed interacts with Orai1 at the preformed ER–PM junctions (12, 13). research; J.S.W., S.S., M.N., P.P., H.T., and Y.G. contributed new reagents/analytic tools; STIM1 uses two major mechanisms to translocate into the ER– J.S.W., S.S., and Y.G. analyzed data; and S.S. and Y.G. wrote the paper. PM junctions: by interactions with phosphatidylinositol-4,5- The authors declare no conflict of interest. This article is a PNAS Direct Submission. bisphosphate (PIP2) in the PM via its C-terminal polybasic res- idues and by interaction with Orai1 or the ER-resident junctate 1J.S.W. and S.S. contributed equally to this work. proteins (14, 15). Recently, septin filaments were shown to play a 2To whom correspondence should be addressed. Email: [email protected]. – role in PIP2 enrichment at the ER PM junctions before STIM1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. recruitment (16). Subsequently, membrane-tethering VAP and 1073/pnas.1524229113/-/DCSupplemental. 2762–2767 | PNAS | March 8, 2016 | vol. 113 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1524229113 Downloaded by guest on September 27, 2021 + effect in recruiting STIM1 to the junctions. Therefore, our of the sarcoplasmic/endoplasmic reticulum Ca2 ATPase (SERCA) studies identify a PIP -independent, but protein interaction- pump or active depletion using anti-CD3 antibody cross-linking 2 + mediated, mechanism by which the junctate–JP4 complex re- (Fig. 1C). Interestingly, ER Ca2 content was also reduced in + cruits STIM1 into the ER–PM junctions to maintain ER Ca2 thapsigargin-treated JP4-depleted cells. We confirmed this obser- vation using a strong ionophore, ionomycin, to ensure complete homeostasis and activate SOCE in T cells. + depletion of the ER Ca2 stores (Fig. 1D). The specific role of JP4 + Results in ER Ca2 content and SOCE reduction was validated by rescue JP4 Plays an Important Role in ER Ca2+ Homeostasis and SOCE in T of these phenotypes in JP4-depleted cells expressing siRNA- – resistant cDNA (Fig. 1E). Together, these results suggested an Cells. To identify components of the ER PM junctions in T cells, 2+ we examined transcript expression of various molecules, including important role of JP4 in ER Ca homeostasis and SOCE in T cells. junctional proteins in excitable cells (calumin, MG29, and JP1 to -4), 2+ proteins involved in lipid modification and transfer (VAP-A, -B, JP4 Deficiency Affects ER Ca Homeostasis, SOCE, and Cytokine and -C and TMEM16A and -B), and membrane-tethering pro- Production in Primary T Cells. Next, we examined the physiologi- teins (E-Syt1, E-Syt2, and E-Syt3) in resting and stimulated Jurkat cal role of JP4 in T cells isolated from JP4 knockout mice (23). As expected, JP4 expression was abrogated in JP4 knockout cells, a leukemic T-cell line. Among these candidates, mRNA ex- + primary naive CD4 T cells (Fig. 2A). Furthermore, we observed pression of JP4 was induced by stimulation in both Jurkat and a significant reduction in SOCE in JP4-deficent naive T cells murine primary T cells (Fig. 1A and Fig. S1A). Increased mRNA after passive store depletion or TCR cross-linking (Fig. 2 B and expression of JP3 was also observed, but only in Jurkat cells. Con- C). Consistent with Jurkat cells, JP4 knockout primary T cells sistent with these mRNA analyses, expression of JP4 protein in 2+ B B also showed a significant reduction in ER Ca content (Fig. 2 ). Jurkat cells was induced after stimulation (Fig. S1 ). JP4 is the least In addition, JP4-deficient effector T cells differentiated under understood member of the junctophilin family and, together with nonpolarizing conditions also showed diminished SOCE after JP3, is known to play an important role in neurons. Although JP1 TCR cross-linking (Fig. 2D). These results confirm an important + and JP2 have specific roles in skeletal and cardiac muscle cells, role of JP4 in ER Ca2 homeostasis and SOCE.