OSBP-Related 5L Maintains Intracellular IP 3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP 2 Hydrolysis This information is current as of September 28, 2021. Mengyang Xu, Biying Zhu, Xiuye Cao, Shannai Li, Dan Li, Huihao Zhou, Vesa M. Olkkonen, Wenbin Zhong, Jun Xu and Daoguang Yan J Immunol published online 17 January 2020 http://www.jimmunol.org/content/early/2020/01/16/jimmun Downloaded from ol.1900671

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 17, 2020, doi:10.4049/jimmunol.1900671 The Journal of Immunology

OSBP-Related Protein 5L Maintains Intracellular 2+ IP3/Ca Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis

Mengyang Xu,*,†,1 Biying Zhu,*,1 Xiuye Cao,*,1 Shannai Li,* Dan Li,* Huihao Zhou,† Vesa M. Olkkonen,‡,x Wenbin Zhong,* Jun Xu,† and Daoguang Yan*

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP2 into the second messengers IP3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCg1 in T cells, extracts PIP2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCg1 (enabling IP3 generation), and eventually maintains intracellular Ca2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca2+-activated

NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCg1 Downloaded from catalysis and its crucial role in human T cell proliferation. The Journal of Immunology, 2020, 204: 000–000.

he second messenger inositol-1,4,5-trisphosphate (IP3) isoforms in T cells (2). Upon anti-CD3 stimulation, linker of ac- is generated by hydrolysis of phosphatidylinositol-4,5- tivated T cells is phosphorylated and subsequently binds and ac- T bisphosphate (PIP2) located in the plasma membrane tivates PLCg1 to cleave PIP2 in a G protein–independent manner (PM), by phospholipase C (PLC) (1). So far, six mammalian (7), whereas in malignant transformed T cell acute lymphoblastic http://www.jimmunol.org/ PLC isozymes have been characterized at the cDNA level, the leukemia (T-ALL) cells, CD3 signaling is swapped in a domi- isozymes differing from each other in tissue distribution, in- nant manner to G protein–/PLCb3-mediated IP3 generation (8). tracellular localization, regulatory mechanisms, or downstream The binding of IP3 to receptors in the endoplasmic reticulum functions (2–4). When a ligand binds to a receptor coupled to a Gq (ER) results in ER Ca2+ egress and mediates a range of cellular heterotrimeric G protein, the a-subunit of Gq induces activity of responses (9). 2+ PLCb, resulting in the cleavage of PIP2 into IP3 and DAG (5). In Ca is a versatile second messenger with a wide range of a receptor tyrosine kinase pathway, the isozyme PLCg1 becomes central physiological roles in processes such as cell growth or phosphorylated upon activation of receptor tyrosine kinase, proliferation, including immune cell responses (10). One response

2+ by guest on September 28, 2021 allowing it to cleave PIP2 (6). PLCg1 is the predominant PLC to the release of Ca through IP3R is enhanced NFAT activity through the Ca2+/calmodulin/calcineurin pathway as a conse- quence of signal transduction (11–13). The NFAT family contains *Department of Biology, Jinan University, Guangzhou 510632, China; †Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen Univer- five members, including four calcium-responsive isoforms named sity, Guangzhou 510006, China; ‡Minerva Foundation Institute for Medical Research, x NFAT1, NFAT2, NFAT3, and NFAT4, and a tonicity-responsive Biomedicum 2U, Helsinki 00290, Finland; and Department of Anatomy, Faculty of enhancer-binding protein (TonEBP, also known as NFAT5) (11). Medicine, University of Helsinki, Helsinki 00014, Finland Calcium-associated NFAT isoforms are usually activated by in- 1M.X., B.Z., and X.C. contributed equally. creased intracellular Ca2+ levels and subsequently translocated ORCIDs: 0000-0002-9675-5007 (H.Z.); 0000-0002-1075-0337 (J.X.). into the nucleus. Once in the nucleus, NFAT1–NFAT4 activate Received for publication June 19, 2019. Accepted for publication December 17, transcription of downstream target with multiple regulatory 2019. roles in cell fate determination, thus directly linking Ca2+ sig- This work was supported by grants from the National Natural Science Foundation of China (81770438, 91439122 [to D.Y.], 81473138 [to J.X.]), the Major Research naling to expression (12). There is evidence that NFAT3 is Program of Guangdong Science and Technology (2017A030308002 [to D.Y.] not expressed in normal T cells (11), although NFAT2 mediates and 2015B010109004 [to J.X.]), the Academy of Finland (285223 and 322647 [to the proliferation of these cells regulated by Ca2+ signaling (11, 12, V.M.O.]), and the Sigrid Juselius Foundation (to V.M.O.). 14, 15). NFAT2 are dephosphorylated by calcineurin M.X., B.Z., and V.M.O. wrote the manuscript. X.C., S.L., D.L., and H.Z. performed 2+ experiments. W.Z., J.X., and D.Y. designed experiments and interpreted the data. activated by Ca , which leads to their nuclear translocation and Address correspondence and reprint requests to Dr. Wenbin Zhong and the induction of NFAT2-mediated gene transcription, further Dr. Daoguang Yan, Department of Biology, Jinan University, Guangzhou activating T cells (11). An NFAT-specific inhibitor, 11R-VIVIT, 510632, China or Dr. Jun Xu, Research Center for Drug Discovery, School of decreases T cells’ proliferation capacity by inhibiting nuclear Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China. E-mail addresses: [email protected] (W.Z.), [email protected] (D.Y.), or translocation of NFAT2 (14, 16–18). [email protected] (J.X.) Oxysterol-binding protein (OSBP) and its relative, oxysterol- The online version of this article contains supplemental material. related protein (ORP), have emerged as mediators of the inter- Abbreviations used in this article: Co-IP, coimmunoprecipitation; ECB, extracellular organelle transfer of cholesterol or phospholipids in exchange calcium buffer; ER, endoplasmic reticulum; IP3, inositol-1,4,5-trisphosphate; MD, for phosphatidylinositol-4-phosphate (PI[4]P) (19, 20). OSBP molecular dynamics; MOE, Molecular Operating Environment; ORP, oxysterol-related protein; OSBP, oxysterol-binding protein; PDB, ; PH, pleckstrin mediates sterol/PI(4)P exchange between the ER and Golgi (19), homology; PI(4)P, phosphatidylinositol-4-phosphate; PIP2, phosphatidylinositol- whereas ORP5/8 exchange phosphatidylserine (PS) for PI(4)P 4,5-bisphosphate; PLC, phospholipase C; PM, plasma membrane; siRNA, small at ER-PM junctions (21, 22). An important role of ORP5/8 as a interfering RNA; T-ALL, T cell acute lymphoblastic leukemia. transporter for PIP2 rather than PI(4)P has recently been explored Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 (23). ORP2 is involved in PIP2 and cholesterol metabolism (24).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900671 2 ORP5L TRANSFERS PIP2 TO PLCg1 FOR IP3 GENERATION

ORP4L is expressed in T-ALL cells, but not in normal T cells, and control gene (actin) expression levels were assessed. PCR primer sets were is essential for cell survival by regulating the activation of PLCb3, designed to amplify specific NFAT1–NFAT4 isoforms and b-actin: NFAT1 a PLC isozyme with a predominant role in IP production in forward, 59-GGGAAAACTGTCACCACCAC-39 and reverse, 59-TTGCTG- 3 TCCATCTGTGGTCT-39 with a product of 420 bp; NFAT2 forward, 59- T-ALL cells (8, 25). Our recent work indicated that ORP4L ex- AGTCCTGGAGATCCCACTCC-39 and reverse, 59-GAATTCGGCTTGCA- tracts and presents PIP2 from the PM for PLCb3 catalysis in CAGGTC-39 with a product of 420 bp; NFAT3 forward, 59-TGGCACT- leukemia stem cells, and thus ORP4L may serve as a target for GACATGGACTTCT-39 and reverse, 59-TGATCTCACTCACTTCCTCCAG- elimination of these malignant cells (26). As ORP5L is involved in 39 with a product of 527 bp; NFAT4 forward, 59-CGACGAGCTCGACTT- 2+ CAAAC-39 and reverse, 59-GAAGACTCCCGGTAGGATGG-39 with a product intracellular Ca signaling and mediates the proliferation of HeLa of 456 bp; b-actin forward, 59-ACGTTGCTATCCAGGCTGTG-39 and re- cells (27), we considered that ORP5L may contribute to the pro- verse, 59-GAGGGCATACCCCTCGTAGA-39 with a product of 116 bp, re- liferation of normal T cells by regulating Ca2+ signaling these cells. spectively. PCR conditions were optimized (35 cycles), and the products were separated by 1% (W/V) agarose gel electrophoresis and visualized with In this study, we report that ORP5L extracts PIP2 from the PM 2+ GelStain (TransGen Biotech). and presents it to PLCg1 for IP3/Ca signaling and thus maintains T cell proliferation. PM preparations A total of 3.5 3 107 cells were collected and centrifuged at 200 3 g for Materials and Methods 5 min. After washing once with cold PBS, cells were subjected to PM Reagents and Abs preparation by using Plasma Membrane Protein Isolation and Cell Frac- tionation Kit (Invent Biotechnologies) according to the manufacturer’s Fluo4-AM, Alexa Fluor-543 goat anti-rabbit IgG, Alexa Fluor-647 rabbit instructions. anti-goat IgG, and TRIzol reagent were purchased from Invitrogen (Carlsbad, CA). Goat anti-ORP5L was purchased from Abcam. Anti–pan-cadherin and Flow cytometry Downloaded from anti-PIP for dot plot were from Santa Cruz Biotechnology (Santa Cruz, CA). 2 5 Anti-PLCg1 was obtained from Cell Signaling (Beverly, MA), and actin was After transfection for 24 h, 5 3 10 cells were stimulated with or without obtained from Proteintech Group (Chicago, IL). Anti-CD3, anti-CD28, and anti-CD3 and anti-CD28 in the presence or absence of 15 mM BAPTA-AM anti-Ki67 were purchased from BioLegend. Anti-PIP2 (ADI-915-062-100) or 1mM 11R-VIVIT in coated 24-well plates for another 24 h. Cells were for coimmunoprecipitation (Co-IP) was obtained from Enzo. BAPTA-AM collected and fixed in 70% ethanol at 220˚C for 1 h. After three washes was purchased from Selleck and 11R-VIVIT from Sigma-Aldrich. siRNA with a cell-staining buffer (BioLegend), cells were stained with anti-Ki67 for 30 min at 37˚C, followed by analysis of the fluorescence by flow against PLCg1. (sc-29452) were purchased from Santa Cruz Biotechnology. http://www.jimmunol.org/ cytometry (Accuri C6; BD Biosciences, Franklin Lakes, NJ). Normal T cell isolation and stimulation Immunofluorescence microscopy A six-well plate was coated with 1 mg/ml anti-CD3 and 2 mg/ml anti-CD28 in 1 ml PBS for 24 h at 4˚C. Fresh peripheral blood cells were obtained T cells were seeded onto coverslips and fixed with 4% paraformaldehyde from healthy volunteer donors. Lymphocytes were separated for 30 min at room temperature, followed by permeabilization with 0.1% using lymphocyte separation medium density gradient (equilibrium) cen- Triton X-100 for 5 min, and blocked with 10% FBS for 30 min at room trifugation (Dakewe Biotech) according to the manufacturer’s instructions. temperature. Cells were then incubated with primary Abs in 5% FBS at 4˚C T cells were further purified from the lymphocytes by using Enhanced overnight. After three washes with PBS (15 min each), cells were incu- Human T Cell Recovery Column Kit (Cedarlane Laboratories, Ontario, bated with fluorophore-conjugated secondary Ab at 37˚C for 30 min. The Canada) according to the manufacturer’s instructions. A total of 5 3 106 specimens were analyzed using Olympus FV3000 Confocal Microscope. by guest on September 28, 2021 T cells suspended in 1 ml of IMDM with penicillin-streptomycin plus L-glycine and 15% FBS were seeded into the coated plate. Cells stimulated Protein purification and detection with or without 1 mg/ml anti-CD3 and 2 mg/ml anti-CD28 for 3 d were After induction at 18˚C for 16 h, Escherichia coli Rosetta (DE3) cells were subjected to transfection. collected by centrifugation, followed by an in-cell lysis buffer (150 mM Gene and small interference RNA transfer NaCl, 1% Triton X-100, 50 mM Tris-HCl [pH 8]) containing 1 mM PMSF at 4˚C for 20 min. Subsequently, the samples were sonicated in a mixture A total of 1 3 106 T cells stimulated with or without 1 mg/ml anti-CD3 and of ice water with the following settings: 4 s on, 6 s off, 60% input, 4 min, 2 mg/ml anti-CD28 for 3 d were transfected with plasmids or small in- and centrifuged (10,000 3 g for 20 min) to obtain total protein extracts. terfering RNAs (siRNAs) by electroporation using a 4D-Nucleofector Glutathione Sepharose Beads (GE Healthcare) were added into the protein System (Lonza, Basel, Switzerland) according to the manufacturer’s in- extracts and incubated overnight at 4˚C. After washing the beads three structions. For gene transfection, cells were transfected with 1 mgof times with cell lysis buffer and 50 mM Tris-HCl (pH 8), the fusion proteins plasmids for 36 h. For siRNA transfection, cells were transfected with 25 were eluted with reduced glutathione elution buffer (20 mM glutathione in pmol of siRNAs plus 1 mg of plasmids for 72 h. The transfection efficiency 50 mM Tris-HCl [pH 8]) and dialyzed for 12 h against PBS at 4˚C. The was verified by Western blotting. The siRNA against ORP5L sequence was purified proteins were stained by Coomassie Brilliant Blue R-250 after 59-CCCTGCCCAGCAGCTACCTGATCTT-39. The siPLCg1 was purchased SDS-PAGE. from Santa Cruz Biotechnology (sc-29452). PIP2 extraction assay Quantitative RT-PCR 7 A total of 3.5 3 10 cells were incubated with 1 mM PIP2 (Sigma-Aldrich) Total RNA was isolated with TRIzol Reagent (Invitrogen) according to the for 10 min at room temperature in extracellular calcium buffer (ECB; manufacturer’s instructions. RNA samples were reverse transcribed using 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 25 mM HEPES random hexamer primers in the presence of RNase Inhibitor (Takara Bio, [pH 7.5], 1 mg/ml BSA, and 5 mM glucose) before PM preparation. Five Shiga, Japan). Quantitative RT-PCR was performed with SYBR Premex micrograms of GST-tag proteins were mixed with 10 mg of PMs in 50 ml EX Taq (Takara Bio) using the CFX96 Sequence Detection System (Bio- of assay buffer (50 mM HEPES [pH 7], 100 mM KCl, 6 mM MgCl2, Rad Laboratories, Hercules, CA). A relative quantification analysis was 0.6 mM CaCl2, 2 mM EGTA) (28). After 30 min incubation at 37˚C, PMs performed using the DD cycle threshold method, with actin as endogenous were collected by centrifugation, and the pellet was resuspended in 20 ml reference. Relative is presented as log (22DD cycle threshold). of lysis buffer (50 mM Tris, [pH 7.5], 300 mM NaCl, 5 mM EGTA, 20 mM The primer sequences were as follows: IL-2 forward, 59-AACTCACCAG- DTT, 2% Triton X-100, and 50 mM NaF) at 37˚C for 30 min. The PIP2 GATGCTCACA-39; IL-2 reverse, 59-CAATGGTTGCTGTCTCATCAGC-39; remaining in the membranes was analyzed by dot-blot. Glutathione actin forward, 59-ACGTTGCTATCCAGGCTGTG-39;andactin reverse, Sepharose Beads were added into the suspension and incubated for 1 h at 59-GAGGGCATACCCCTCGTAGA-39. room temperature to pull down the proteins and their bound PIP2,fol- lowed by centrifugation and analysis of the PIP2 remaining in the pellet RT-PCR by dot-blot. Total RNA was isolated with TRIzol Reagent (Invitrogen) according to the Reconstituted cell-free system manufacturer’s instructions. RNA samples were reverse transcribed with PrimeScript TM RT Reagent Kit (TaKaRa Bio) according to the manu- As described previously, cells were incubated with 1 mM PIP2 (Sigma- facturer’s instructions. The NFAT genes (NFAT1–NFAT4) and the internal Aldrich) for 10 min at room temperature in ECB before PM preparation. The Journal of Immunology 3

Five micrograms of GST-tag proteins were mixed with 10 mg of PMs in the the final results, where F0 represents baseline fluorescence intensity in 50 ml of assay buffer (50 mM HEPES [pH 7], 100 mM KCl, 6 mM MgCl2, each cell. 0.6 mM CaCl2, 2 mM EGTA). The assay mixture was incubated on ice for 10 min, and the reaction was started by incubation at 37˚C for 10 min. Western blot analysis After the reaction, the PMs were collected by centrifugation, and the PIP 2 Cells were harvested and lysed with lysis buffer (50 mM Tris-Cl [pH 8], and DAG remaining in the pellet were analyzed. The suspension was used 150 mM NaCl, 0.5 mM MgCl , 10% glycerol, 1% Triton X-100, 0.1% for IP measurements. 2 3 SDS) with protease inhibitor mixture (Roche Diagnostics) on ice for 30 min. Samples were centrifuged at 4˚C for 10 min at 12,000 3 g. The Dot blots for PIP2 levels supernatants were collected, and protein concentrations were measured by The PIP2 in PMs was released by incubation of lysis buffer (50 mM Tris the BCA Protein Assay Kit (Beyotime) according to the manufacturer’s [pH 7.5], 300 mM NaCl, 5 mM EGTA, 20 mM DTT, 2% Triton X-100, and instructions. Protein extracts were run on a 10 or 12% SDS polyacrylamide 50 mM NaF) at 37˚C for 30 min. Dot blots were conducted as described gel before they were transferred to a polyvinylidene difluoride membrane. (29), and the following details were performed in accordance with our Membranes were blocked with 5% milk for 1 h and incubated with pri- previous work (26). Briefly, after centrifugation, 1 ml of suspension was mary Abs overnight, followed by incubation with the secondary Abs for spotted onto nitrocellulose membrane (Bio-Rad Laboratories), probed with 1 h at room temperature. Primary Abs used in this study were as follows: PIP2 Ab (2C11) (1:50, sc-53412; Santa Cruz Biotechnology) or PM in- anti-ORP5L (1:1000; catalog no. ab59016; Abcam), anti-actin (1:3000; ternal loading control pan-cadherin Ab (C-19) (1:500, sc-1499; Santa Cruz catalog no. 60008-1-Ig; Proteintech Group), and anti-PLCg1 (1:1000; Biotechnology), and detected using an HRP-conjugated secondary Ab and catalog no. 2822; Cell Signaling Technology). ECL. Experiments were repeated at least three times. Co-IP IP3 and DAG measurement Cells were lysed in Co-IP lysis buffer (150 mM NaCl, 0.5% [v/v] Triton X-

6 Downloaded from A total of 6 3 10 cells were collected and centrifuged at 200 3 g for 100, 10 mM MgCl2, 50 mM Tris-HCl [pH 8], 10% glycerol [v/v]) with 5 min. After washing once with cold PBS, cells were lysed with 240 mlof protein inhibitor mixture (Roche Diagnostics) and 1 mM PMSF at 4˚C for lysis buffer (150 mM NaCl, 0.5% [v/v] Triton X-100, 10 mM MgCl2, 1 h, followed by centrifuging at 12,000 3 g at 4˚C for 10 min. The su- 50 mM Tris-HCl [pH 8], 10% glycerol [v/v]) at 4˚C for 30 min. The IP3 pernatant was preadsorbed with protein A/G agarose (Invitrogen) for 1 h, level was measured using the HitHunter IP3 Fluorescence Polarization and the primary Ab (1:50) was added into the supernatant and incubated Assay Kit (DiscoverRx Tech, Fremont, CA) according to the manufac- overnight at 4˚C. After three washes with lysis buffer, the bound proteins turer’s instructions. For the DAG measurement, the PMs were disrupted by were eluted into SDS loading buffer by incubation at 99˚C for 10 min. The ultrasonication, and after centrifugation, the suspension was used for coimmunoprecipitated proteins were analyzed by Western blot. http://www.jimmunol.org/ analysis by using the (DAG/DG) ELISA Kit (J&L Biological, Shanghai, China) according to the manufacturer’s instructions. GST pull-down Homology modeling and docking simulation Equal amounts of GST, GST–wild-type ORP5L, and GST-ORP5L (Mut4) fusion proteins were immobilized with Glutathione Sepharose 4B. Cells The sequences of ORP5L and PLCg1 were downloaded from the GenBank were lysed in Co-IP lysis buffer (150 mM NaCl, 0.5% [v/v] Triton X-100, (entry: AAH32646.2; AAI36467.1). The template for sequence alignment 10 mM MgCl2, 50 mM Tris-HCl [pH 8], 10% glycerol [v/v]) containing was identified through the Molecular Operating Environment (MOE)- protein inhibitor mixture (Roche Diagnostics) and 1 mM PMSF at 4˚C Search Protein Data Bank (PDB) module (30). The three-dimensional for 1 h, followed by centrifugation at 12,000 3 g at 4˚C for 10 min. structure of OSBP homology 6 was downloaded from PDB (PDB code: GST–wild-type ORP5L–coupled Sepharose or GST-ORP5L (Mut4)–cou- 4ph7, http://www.rcsb.org/pdb/home/home.do) as the template structure of pled Sepharose was added into the supernatant and incubated overnight at by guest on September 28, 2021 ORP5L-ORD domain, and the three-dimensional structure of OSBP- 4˚C. After three washes with lysis buffer, the bound proteins were eluted related protein 8 (PDB accession code: 5U77) was downloaded as the into SDS loading buffer by boiling at 99˚C for 10 min, followed by template structure of ORP5L–pleckstrin homology (PH) domain. The analysis by Western blot. three-dimensional structure of 1-phosphatidylinositol 4, 5-bisphosphate phosphodiesterase b-3 was downloaded from PDB (PDB accession code: Circular dichroism spectroscopy analysis 4gnk) as the template structure of PLCg1. The homology models of ORP5L and PLCg1 were built with MOE 2015.10 and structural optimi- Protein folding and stability studies were conducted on a Jasco-810 zation was carried out by molecular dynamics (MD) simulation. spectropolarimeter equipped with a Peltier temperature controller. Sam- The AMBER 12 Software was used to perform the MD simulations (31). ples were composed of 0.5 mg/ml protein in 100 mM Tris-HCl (pH 7.4). The Amber ff03.R1 (32) force field was applied to the proteins. Topology Spectral scans were performed from 180 to 250 nm with a step resolution and parameter files were generated using the LEaP program (which con- of 1 nm and bandwidth of 1 nm. A 0.1-cm pathlength quartz cuvette was structs biopolymers from the component residues, solvates the system, and used for the measurements, and values from scans were averaged for each prepares lists of force field terms and their associated parameters). (31). sample. Solvent background was subtracted from each sample. Protein Homology models’ molecules were solvated with TIP3P water model in a stability was monitored at 220 nm, and data were collected at 5˚C intervals cubic periodic boundary box to generate required systems for MD simu- as the sample temperature was increased from 20 to 80˚C at a rate of lations, and systems were neutralized using appropriate number of coun- 2˚C per minute. terions. The neutralized system was then minimized, heated up to 300 K Quantification and statistical analysis temperature, downloaded, and equilibrated until the pressure and energies of systems were stabilized. Finally, equilibrated systems were used to run All data are presented as mean 6 SD. All experiments were repeated at 10 ns MD simulations. least three times. All comparisons between groups were made by unpaired Modeling and simulations to model the ORP5L and PLCg1 complex and two-tailed Student t test. Differences with *p , 0.05, **p , 0.01, and to predict the key residues involved in the interaction were performed ***p , 0.001 were considered statistically significant. using MOE 2015.10. The best docking results were selected according to the lowest total score. Results 2+ Measurements of Ca efflux ORP5L interacts with PLCg1 in T cells 5 Cells (1 3 10 cells) resuspended in 100 ml ECB (130 mM NaCl, 5 mM Our previous observation of the novel role that ORP4L plays in KCl, 1.5 mM CaCl2, 1 mM MgCl2, 25 mM HEPES [pH 7.5], 1 mg/ml PIP extraction (26) prompted us to investigate whether other BSA, and 5 mM glucose) were loaded with 1 mM Fluo-4 AM for 30 min 2 at 37˚C, followed by an additional 30 min of incubation at 37˚C to permit ORPs could execute a similar function. PLCg1 is the predominant dye de-esterification. Culture dishes were mounted on the stage of an isoform responsible for TCR signaling in T cells; its knockdown 2+ inverted confocal microscope (Olympus FV3000) equipped with a decreased IP3 production and Ca peak amplitude (Supplemental 1003 oil objective. Cells were excited by low-intensity 488-nm laser Fig. 1A, 1B). As ORP5L has recently been reported to regulate the excitation, and images were acquired at 2 s intervals under time-lapse mode. Cells were stimulated with the 10 mg/ml anti-CD3 Ab to measure level of PIP2 in the PM (23), we investigated the putative interaction Ca2+ efflux. Image data were subsequently analyzed using ImageJ of ORP5L and PLCg1. We observed that ORP5L is expressed in (National Institutes of Health) and were presented as a ratio of F/F0 in normal T cells and interacts with PLCg1, as evidenced by Co-IP 4 ORP5L TRANSFERS PIP2 TO PLCg1 FOR IP3 GENERATION

FIGURE 1. ORP5L interacts with PLCg1. (A) Co-IP of ORP5L and PLCg1 in T cells. (B) GST pull-down analysis of ORP5L and PLCg1in T cells. (C) Representative confocal images show-

ing the colocalization of ORP5L and PLCg1in Downloaded from T cells. Scale bar, 10 mm. (D) ORP5L interacts with PLCg1 as modeled by computer docking, the interacting amino acids of ORP5L (cyan) and PLCg1 (gold) are shown. (E) GST pull-down interaction analysis of ORP5L (Mut4) and PLCg1 in T cells. http://www.jimmunol.org/ by guest on September 28, 2021

(Fig. 1A) and GST pull-down (Fig. 1B) experiments. Consistently, models shows a markedly lower energy (Table II, total energy confocal immunofluorescence microscopy revealed an extensive 215.02 kcal/mol) than the other models. The total energy of colocalization of the two proteins in the PM region of T cells ORP5L-PH/PLCg1 complex homology model (Table I, total en- (Fig. 1C). In addition to the ORP5-ORD, both the ORP5L-PH ergy 23.26 kcal/mol), ORP5L-ORD/PLCg1-SH2 (Table III, total domain and PLCg1 can bind PIP2. Although we did not in this energy 22.17 kcal/mol), and ORP5L-PH/PLCg1-SH2 (Table IV, study address in detail the complex of ORP5/PLCg1/PIP2,we total energy 24.57 kcal/mol) were higher than the complex of hypothesize that the binding of ORP5L-PH domain and PLCg1to ORP5L-ORD/PLCg1 homology model. These data suggest that PIP2 could facilitate the interaction of the two proteins. the ORP5L-ORD prefers to bind PLCg1, and the corresponding To further analyze how ORP5L binds to PLCg1, protein-protein complex model was selected for the protein-protein binding mode docking was performed with MOE 2015.10 (30). The ORP5L-PH analysis. The complex model of ORP5L (cyan) and PLCg1 (gold) domain and ORP5L-ORD domain were docked with the PLCg1 interaction were visualized (Supplemental Video 1). On the basis homology model (without SH2 domain) (Tables I and II) and the of in silico docking studies, we propose that the interaction of PLCg1-SH2 domain. The low-energy regions consistently corre- ORP5L with PLCg1 occurs via two ionic bonds and four H-bonds. sponded to actual binding sites (33). The best docking result was E361 and D606 of ORP5L formed the ionic bonds with K42 and selected according to the lowest total score (Supplemental R58 of PLCg1. K439 and G355 of ORP5L formed H-bonds with Table I). The complex of ORP5L-ORD and PLCg1 homology Y93, E95, and D96 of PLCg1, respectively (Table II), suggesting

Table I. Analysis of the ORP5L-PH and PLCg1-model docking results

Type Chain A Set A Chain B Set B Energy (kal/mol) Distance (A)˚ Ionic bond ORP5L K140 PLCg1 E290 22.250 3.034 Hydrogen bond ORP5L K189 PLCg1 P291 20.173 2.481 Hydrogen bond ORP5L D195 PLCg1 T300 20.834 2.634 Contacts between residues of ORP5L-PH and PLCg1-model. The Journal of Immunology 5

Table II. Analysis of the ORP5L-ORD and PLCg1-model docking results

Type Chain A Set A Chain B Set B Energy (kal/mol) Distance (A)˚ Ionic bond ORP5L E361 PLCg1 K42 20.531 3.289 Ionic bond ORP5L D606 PLCg1 R58 26.642 3.59 Hydrogen bond ORP5L K439 PLCg1 Y93 21.4 3.061 Hydrogen bond ORP5L G355 PLCg1 E95 24.044 3.186 Hydrogen bond ORP5L G355 PLCg1 D96 22.4 3.074 Contacts between residues of ORP5L-ORD and PLCg1-model.

that PLCg1 could interact with ORP5L via E361, D606, K439, and PIP2 and generation of second messengers in the presence of wild- G355 (Fig. 1D). We next generated a mutant ORP5L with all these type ORP5L but not the mutants (Fig. 3B). four residues replaced (G355P, E361G, K439G, D606G, designated To bypass the effects of endogenous PLCg1 in this system, ORP5L [Mut4]) to experimentally validate the in silico docking we prepared PMs from T cells subjected to PLCg1 knockdown analysis. Upon the results that ORP5L (Mut4) protein is prop- (with above 80% knockdown efficiency, shown in Supplemental erly folded and is as stable as wild-type ORP5L (Supplemental Fig. 2D, bottom) for cell-free experiments. We found that ORP5L Fig. 2A–C), we performed GST pull-down assays, demonstrating addition failed to cleave PIP2 and induce DAG/IP3 generation in that the binding of ORP5L (Mut4) to PLCg1 was significantly the system when using PMs isolated from PLCg1 knockdown Downloaded from compromised as compared with wild-type ORP5L (Fig. 1E). T cells, as compared with the PMs isolated from wild-type T cells (Fig. 3C, 3D). Furthermore, when using PMs from ORP5L ORP5L acts as a cofactor for PIP hydrolysis by PLCg1in 2 knockdown T cells, ORP5L (Mut2) and ORP5L (Mut4) proteins normal T cells failed to induce cleavage of PIP2 (Fig. 3C). These results sug- Although PLCb3 hydrolyzes PIP2 for generating IP3 and DAG, it gested that ORP5L enhanced PIP2 cleavage via binding PLCg1 is unable to extract PIP2 from the PM and facilitate catalysis in the and promoting its activation. The above observations established http://www.jimmunol.org/ absence of ORP4L in leukemia stem cells (26). First, we inves- a crucial function of ORP5L in extracting and presenting PIP2 tigated whether PLCg1 could extract PIP2 from the PM. After to PLCg1 to enable IP3/DAG generation in T cells, similar to the incubating PMs with purified PLCg1, the PIP2 remaining in the effect of ORP4L in leukemia stem cells (23). PM was analyzed to determine the extraction capacity of the ORP5L maintains Ca2+ signaling in T cells proteins. We found that PLCg1 alone was unable to extract PIP2 (Fig. 2A). In contrast, incubation with ORP5L decreased the We next studied the PIP2 regulation in the PM of living T cells. amount of PIP2 in the PM fraction, with a concomitant increase of Overexpression of the wild-type and mutant ORP5L proteins was its amount bound to the ORP5L (Fig. 2B), demonstrating the monitored by Western blot assay (Supplemental Fig. 2D, upper). extraction of PIP2 from PM by ORP5L. Furthermore, the ORD We then measured PIP2 level in the PM, DAG/IP3 production, and domain of ORP5L is crucial for its extraction capacity, whereas Ca2+ release. The results showed that wild-type ORP5L significantly by guest on September 28, 2021 the PH domain alone could not carry out the extraction (Fig. 2C). increased the PIP2 clearance upon anti-CD3 stimulation. However, We next reconstituted a cell-free system to study the hydrolysis the ORP5L (Mut2) and ORP5L (Mut4) mutants were unable do this of PIP2 in the presence or absence of ORP5L and PLCg1. We (Fig. 4A). In addition, overexpression of wild-type ORP5L, but not generated a mutant ORP5L (H478A, H479A), designated ORP5L the mutants, promoted DAG and IP3 production (Fig. 4B). 2+ (Mut2), deficient in PIP2 binding (23) and confirmed the proper IP3 acts as second messenger with a key role in Ca signaling. folding and stability of the ORP5L (Mut2) protein (Supplemental ORP5L, but not ORP8, was reported to mediate Ca2+ signaling in Fig. 2A–C). After incubations with the purified proteins, pellets HeLa cells, promoting their proliferation capacity (27). We further 2+ containing the PMs were analyzed for PIP2 and DAG, and the investigated the role of ORP5L in ER Ca release of T cells upon supernatants were analyzed for IP3. PLCg1 alone did not induce anti-CD3 stimulation. In agreement with the observed IP3 pro- 2+ loss of PIP2. However, combination of ORP5L and PLCg1dra- duction, wild-type ORP5L enhanced the [Ca ]i peak amplitude, matically decreased the PIP2 in the membranes. Importantly, whereas the ORP5L mutants defective in PIP2 or PLCg1 binding ORP5L mutants deficient in PIP2 or PLCg1 binding (ORP5L- lacked this activity (Fig. 4C). Mut2 and ORP5L-Mut4, respectively) abolished these effects To further verify the concerted action of ORP5L and PLCg1in 2+ (Fig. 3A). ORP5L overexpression promoted PIP2 binding to Ca signaling, we combined manipulation ORP5L and PLCg1in PLCg1, but the ORP5L (Mut2) and ORP5L (Mut4) lacked this living T cells. ORP5L overexpression promoted PIP2 clearance effect (Supplemental Fig. 3A, 3B), suggesting that the hydrolysis from the PM, but the influence was abolished in PLCg1-knockdown of PIP2 in the PM requires the presentation of PIP2 to PLCg1 cells. Meanwhile, in ORP5L knockdown cells, the PM PIP2 level by ORP5L. To seek further evidence for this, we measured DAG was the same as that in PLCg1 knockdown cells and higher than and IP3 generation in the PM and soluble fraction, respectively. The in the controls, and overexpression of the ORP5L (Mut2) and results revealed that PLCg1 significantly increased the clearance of ORP5L (Mut4) failed to rescue the effects by ORP5L knockdown

Table III. Analysis of the ORP5L-ORD and PLCg1-SH2 (546–790) docking results

Type Chain A Set A Chain B Set B Energy (kal/mol) Distance (A)˚ Hydrogen bond ORP5L E365 PLCg1 R652 21.385 4.06 Hydrogen bond ORP5L L514 PLCg12 M789 20.528 3.07 Ionic bond ORP5L G545 PLCg1 E655 20.254 3.32

Contacts between residues of ORP5L-ORD and PLCg1-SH2 (546–790). 6 ORP5L TRANSFERS PIP2 TO PLCg1 FOR IP3 GENERATION

Table IV. Analysis of the ORP5L-PH and PLCg1-SH2 (546–790) docking results

Type Chain A Set A Chain B Set B Energy (kal/mol) Distance (A)˚ Hydrogen bond ORP5L K134 PLCg1 Y572 21.050 3.12 Hydrogen bond ORP5L K144 PLCg1 G577 20.192 3.082 Hydrogen bond ORP5L K144 PLCg1 E655 22.372 2.937 Hydrogen bond ORP5L W142 PLCg1 D732 20.960 3.91

Contacts between residues of ORP5L-PH and PLCg1-SH2 (546–790).

(Fig. 4D). Cleavage of PIP2 in PM leads to changes in downstream ORP5L knockdown cells could not be rescued by expressing 2+ signaling molecules, such as DAG/IP3 generation and ER Ca ORP5L (Mut2) or ORP5L (Mut4) (Fig. 4E). The same effects release. Similar to the results in a cell-free system, increased appeared in ER Ca2+ release (Fig. 4F). Taken together, the results DAG/IP3 generation by ORP5L were attenuated in cells with above suggested that the interaction with PLCg1 and PIP2 bind- 2+ PLCg1 knockdown, and the decreased DAG/IP3 generation in ing are required for the function of ORP5L in Ca signaling. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. ORP5L extracts PIP2 from the PM. (A) Dot-blot to determine the ability of PLCg1 to extract PIP2.(B and C) Dot-blot to determine the ability of ORP5L, ORP5L-ORD domain (B), and ORP5L-PH domain (C) to extract PIP2. After the reactions, the PIP2 remaining in the PM and soluble in the buffer were analyzed. Pan-cadherin (p-cad) was used as an internal control of PM and GST as a soluble control protein in the buffer. Quantification of the dot blots indicated the relative PIP2 level; corresponding graphs are shown in the right-hand panel. Data are representative of at least three independent experiments. Mean 6 SD, Student t test. **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. ORP5L presents PIP2 to PLCg1 for catalysis. (A) The amount of PIP2 in the PM after cell-free reconstituted reactions in the presence of the indicated recombinant proteins. The quantification of the dot blots indicates the relative PIP2 level; the corresponding graph is shown in the right-hand panel. (B) The amount of DAG in the PM and the IP3 released after cell-free reconstituted reactions in the presence of the indicated recombinant proteins. (C) The amount of PIP2 in the PM from PLCg1 or ORP5L knockdown cells after cell-free reconstituted reactions in the presence of the indicated recombinant proteins. The quantification of the dot blots indicates the relative PIP2 level; the corresponding graph is shown in the right-hand panel. (D) The amount of DAG in the PM and the IP3 released from PLCg1 or ORP5L knockdown cells after cell-free reconstituted reactions in the presence of the indicated recombinant proteins. Data represent mean 6 SD of at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. 8 ORP5L TRANSFERS PIP2 TO PLCg1 FOR IP3 GENERATION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

2+ FIGURE 4. ORP5L maintains Ca signaling. (A) The amount of PIP2 in the PM preparation from T cells transfected with wild-type ORP5L, ORP5L (Mut2), or ORP5L (Mut4). Pan-cadherin (p-cad) is used as internal control in the PM. The quantification of the dot blots indicates relative PIP2 level; the corresponding graph is shown in the right-hand panel. (B) The amount of DAG in the PM (left) and the IP3 production (right) upon anti-CD3 (10 mg/ml) stimulation for 3 min in the indicated cells. (C)Ca2+ efflux in T cells transfected with the indicated constructs, induced by anti-CD3 (10 mg/ml). (D) The amount of PIP2 in the PM preparation from T cells cotransfected with siNT, siPLCg1 or siORP5L plus Mock, ORP5L (Mut2) or ORP5L (Mut4). P-cad is used as internal control in the PM. The quantification of the dot blots indicates relative PIP2 level; the corresponding graph is shown in the right-hand panel. 2+ (E) The amount of DAG in the PM (left) and the IP3 production (right) upon anti-CD3 (10 mg/ml) stimulation for 3 min in the indicated cells. (F)Ca efflux in T cells transfected with the indicated constructs, induced by anti-CD3 (10 mg/ml). Data represent mean 6 SD of at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test.

However, we cannot completely exclude the possibility that of wild-type ORP5L, but not the ORP5L (Mut2) and (Mut4), ORP5L might also itself be able to cleave PIP2. promoted T cell proliferation upon anti-CD3 and anti-CD28 stim- ulation (Fig. 5A, 5B). When we employed the cell-permeable ORP5L promotes the proliferation of activated T cells in 2+ 2+ calcium chelator BAPTA-AM to deprive the intracellular Ca Ca -dependent manner (34), the increase of proliferation capability prompted by We next investigated whether ORP5L regulates the proliferation of ORP5L was abolished (Fig. 5A, 5B), suggesting that ORP5L 2+ T cells. Similar to the effects on IP3/Ca signaling, overexpression promotes the proliferation of activated T cells in a manner that The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. ORP5L promotes T cell proliferation in a Ca2+-dependent manner. (A) Flow cytometry analysis of Ki67 expression in T cells after transfection and 24 h stimulation with anti-CD3 and anti-CD28 in the presence or absence of 15 mM BAPTA-AM. (B) The corresponding analysis plot. Data are representative of at least three independent experiments. Mean 6 SD, Student t test. **p , 0.01, ***p , 0.001.

depends on its ability to interact with PIP2 and PLCg1andto 6A, 6B), suggesting that ORP5L promotes T cell proliferation control Ca2+ signaling. via NFAT2 activity. IL-2 acts downstream of NFAT2 to mediate the proliferation of T cell proliferation induced by ORP5L requires NFAT2 T cells (36). We therefore quantified IL-2 mRNA by real-time Several ORPs have been implicated in the control of cell prolif- quantitative PCR in ORP5L-overexpressing T cells upon stimu- eration. It has been shown that ORP4 silencing halts cell prolif- lation. The wild-type ORP5L, but not the ORP5L (Mut2) and eration and that ORP4L modulates proliferation through Ca2+ (Mut4), increased IL-2 mRNA expression; the effects were and the NFAT pathway (35). Also, it is well established that in- abolished by NFAT2 inhibition (Fig. 6C). These data provide tracellular Ca2+ plays key roles in controlling cell proliferation compelling evidence that the ORP5L-induced T cell prolifera- via the Ca2+ regulated transcription factor NFAT (27). Although tion is mediated by NFAT2. NFAT1, NFAT2, and NFAT4 are coexpressed in T cells and are activated in response to TCR engagement, NFAT4 expression by Discussion peripheral T cells is low compared with that of NFAT1 or NFAT2 Our previous study showed that the wild-type ORP4L, but not the (11). Our RT-PCR results also confirmed the predominant ex- ORP4L mutants deficient in PIP2 or PLCb3 binding, induced PIP2 pression of NFAT1 and NFAT2 in T cells (Supplemental Fig. 3C). hydrolysis by PLCb3 (26). In this study, we found that ORP5L Given that NFAT1 is constitutively expressed in naive T cells (11), analogously extracts PIP2 from the PM, whereas PLCg1 alone is NFAT2 deficiency in CD8+ T cells increased the threshold of unable to do this, suggesting that ORP5L acts as a crucial cofactor T cell activation and reduced cell proliferation (15); thus, NFAT2 for PLCg1. We further confirmed that ORP5L physically interacts is the putative crucial NFAT isoform mediating the proliferation with PLCg1 in normal T cells. In living T cells, wild-type ORP5L of activated normal T cells. We assessed T cell proliferation overexpression resulted in the decreased retention of PIP2 in the upon treatment with the NFAT2-specific inhibitor 11R-VIVIT PM. However, overexpression of an ORP5L mutant with a defect that inhibits NFAT2 translocation into the nucleus (16). The in PLCg1 binding had no such effect, revealing that the function results demonstrated that the ORP5L-mediated T cell prolifer- of ORP5L in TCR signaling relies on its interaction with PLCg1. ation could be completely abolished by the 11R-VIVIT (Fig. These results indicated that PIP2 extracted by ORPs could be 10 ORP5L TRANSFERS PIP2 TO PLCg1 FOR IP3 GENERATION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. T cell proliferation induced by ORP5L requires NFAT2. (A) Flow cytometry analysis of Ki67 expression in T cells after transfection and 24 h stimulation with anti-CD3 and anti-CD28 in the presence or absence of 1 mM 11R-VIVIT. (B) The corresponding analysis plot. (C) Quantitative RT-PCR analysis of IL-2 mRNA expression upon treatment with or without the NFAT2 inhibitor for 24 h. Data are representative of at least three independent experiments. Mean 6 SD, Student t test. **p , 0.01, ***p , 0.001. delivered to their cognate PLC isoforms via a physical interaction Of note, calcium signaling plays a predominant role in mediating of the proteins. Thus, ORPs may act as cofactors required for PIP2 T cell activation, proliferation, and differentiation (13, 37–39). extraction from the PM and its presentation to PLC catalysis in a TCR signal transduction is considered to represent the initial cell type–specific manner. Additionally, recent evidence showed phase of T cell activation and proliferation (13). Stimulation of that ORP2 also exchanges PIP2 and cholesterol between PM and the TCR by specific Ags, including anti-CD3 and anti-CD28, other cellular organelle membranes, such as ER, Golgi, and [which activates TCR signaling, leading to activation of PLCg1 endosomes (24). These results, to our knowledge, introduce a and its downstream Ca2+ signaling, further promoting cell pro- new concept: the extraction of PIP2 from the PM by ORPs with liferation (40)] is crucial for the immunological functions of implications for a number of PIP2-dependent cellular functions. T cells. However, it is not fully understood how the TCR signal The Journal of Immunology 11 transduction from extracellular to intracellular, which drives T cell 16. Noguchi, H., M. Matsushita, T. Okitsu, A. Moriwaki, K. Tomizawa, S. Kang, S. T. Li, N. Kobayashi, S. Matsumoto, K. Tanaka, et al. 2004. A new cell- proliferation, takes place. 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IP3 [nM ] 1 2 3 4 5 6 IUE1. FIGURE *** shPLC shNT 2+ rnin pnPLC upon transient J 1 PLCγ stepeoiatioomi el.Maueeto anti-CD3- of Measurement cells. T in isoform predominant the is 1 B F/F0 0.5 1.0 1.5 2.0 2.5 3.0 γ 0 1.5 3 4.5 6 7.5 ncdw nnra cells T normal in knockdown 1 Anti-CD3 shPLC shNT Time (min) f J 1 SD, Student’s t test.

. F/F0 aaaerepresentative are Data 1.0 1.5 2.0 2.5 3.0 *** shPLC shNT J 1 induced fat of A

170KD 130KD GST-ORP5L 100KD (127KD) 70KD 55KD 40KD 35KD 25KD

B ORP5L C ORP5L ORP5L(Mut2) ORP5L(Mut2) ORP5L(Mut4) ORP5L(Mut4)

300 0

200 -10

100 -20 0 -30 -100 CD(mdge) CD(mdge) -200 -40

-300 -50 180 200 220 240 20 40 60 80

Wavelength(nm) Temperature(q C)

D

ORP5L (101KD)

Actin (42KD)

ORP5L (101KD)

PLCγ1 (155KD)

Actin (42KD)

Supplemental FIGURE 2. (A) Coomassie brilliant blue R-250 stained SDS-PAGE of wild-type and mutants ORP5L proteins.(B) CD spectroscopy analysis of the folding ability of WT-ORP5L and mutant forms of the protein. (C) CD spectroscopy analysis of the stability of WT-ORP5L and mutant forms of the protein. (D) Western blot analysis of ORP5L expression in T cells transfected with Mock, ORP5L, ORP5L (Mut2) and ORP5L (Mut4), and PLCγ1 expression in T cells subjected to knockdown with siNT, siORP5L or siPLCγ1. Actin acts as the internal control. Co Supplemental NFAT The ORP Agarose representative C (150KD) B A H-chain (48KD) PLCγ1 Imnpeiiainaayi fPLC of analysis -Immunoprecipitation IP: NFAT Lmdl) h orsodn nlssdarmo PLC of diagram analysis corresponding The 5L(middle). (527bp), 3 e lcrpoei mgso xrsinof expression of images electrophoresis gel sfrsmN xrsinwsaaye yR-C,adtePRpout of products PCR the and RT-PCR, by analyzed was expression mRNA isoforms ORP5L (101KD) ORP5L fa es he needn xeiet.*p<00,** .0,Mean 0.001, < ***p 0.01, < **p experiments. independent three least at of IUE3. FIGURE Actin (42KD) NFAT Input 46p and (456bp) 4

Control IgG Mock ( A Anti-PIP2 etr ltvrfigsmlroeepeso eeso TOPLadth and WT-ORP5L of levels overexpression similar verifying blot Western ) ORP5L Control IgG NFAT actin Actin Mock

16p eevsaie ihGlti staining. GelStain with visualized were (116bp) Anti-PIP2 γ n I2i el vrxrsigwl-yeo mutant or wild-type overexpressing cells T in PIP2 and 1 ORP5L (Mut2) FT FT FT NFAT4 NFAT3 NFAT2 NFAT1 Control IgG R5 R5(u2 ORP5L(Mut4) ORP5L(Mut2) ORP5L NFAT Anti-PIP2 ORP5L (Mut4) sfrs( isoforms Control IgG γ nAt-I2imnpeiiae rgtpnl.Data panel). (right immunoprecipitates Anti-PIP2 in 1

Anti-PIP2 NFAT

-)i el sltdfo peripheral from isolated cells T in 1-4) R elative P LC γ1 expression by PIP2 co-immunoprecipitation 0 1 2 3 4 5 *** NFAT f (420bp), 1 SD, ** Student’s ** NFAT emutants (420 2 test. t om of forms blood. .( p , bp) ( are C B ) ) Supplemental Table I. Top 5 template results for the ORP5L

Name Title Identity

4ph7 Oxysterol-binding protein homolog 6 36.44

4b2z Oxysterol-binding protein homolog 6 36.44

5zm5 Oxysterol-binding protein-related protein 1 31.34

5zm8 Oxysterol-binding protein-related protein 2 30.29

5h2d Oxysterol-binding protein-related protein 1 27.46

The percentage sequence identity is calculated from the number of identical residues between ORP5L and templates.

Legend for Supplemental video. Molecular dynamics simulation of ORP5L (cyan) with PLCγ1 (gold). 10 ns molecular dynamics (MD) simulations were performed on the protein-protein complex system. The visual stability of ORP5L-PLCγ1 complex system is shown during the 10 ns simulation of the ORP5L-PLCγ1 complex system.