Synapse CD3 Complex at the Immunological

The CD3 ζ Subunit Contains a Phosphoinositide-Binding Motif That Is Required for the Stable Accumulation of TCR −CD3 Complex at the Immunological This information is current as Synapse of September 24, 2021. Laura M. DeFord-Watts, David S. Dougall, Serkan Belkaya, Blake A. Johnson, Jennifer L. Eitson, Kole T. Roybal, Barbara Barylko, Joseph P. Albanesi, Christoph Wülfing and

Nicolai S. C. van Oers Downloaded from J Immunol 2011; 186:6839-6847; Prepublished online 4 May 2011; doi: 10.4049/jimmunol.1002721 http://www.jimmunol.org/content/186/12/6839 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2011/05/04/jimmunol.100272 Material 1.DC1 References This article cites 43 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/186/12/6839.full#ref-list-1 by guest on September 24, 2021

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The CD3 z Subunit Contains a Phosphoinositide-Binding Motif That Is Required for the Stable Accumulation of TCR–CD3 Complex at the Immunological Synapse

Laura M. DeFord-Watts,* David S. Dougall,* Serkan Belkaya,* Blake A. Johnson,* Jennifer L. Eitson,* Kole T. Roybal,* Barbara Barylko,† Joseph P. Albanesi,† Christoph Wu¨lﬁng,*,‡ and Nicolai S. C. van Oers*,x

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 « subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 « BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 z also contains Downloaded from several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 z to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 z. Instead, the elimination of the phosphoinositide-binding function of CD3 z significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell–APC interactions. Without its phosphoinositide-binding functions, CD3 z was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 z BRS–phosphoinosi- http://www.jimmunol.org/ tide interactions in supporting T cell activation. The Journal of Immunology, 2011, 186: 6839–6847.

he TCR recognizes both self- and foreign peptides em- complexed by the tandem Src-homology 2 (SH2) domains of bedded in the peptide-binding groove of MHC molecules. the Syk/ZAP-70 family of protein tyrosine kinases (4). This piv- T TCR interactions with the appropriate peptide–MHC com- otal phosphotyrosine–SH2 interaction promotes a conformational plexes initiate an intricate series of intracellular signaling events change within a linker region of ZAP-70, thereby enhancing the leading to T cell activation. Although the a and b subunits of phosphorylation of the kinase and creating a catalytically active the TCR are essential for peptide–MHC recognition, intracellular structure (5). Active ZAP-70 proceeds to phosphorylate a number by guest on September 24, 2021 signaling events are mediated by the paired invariant subunits of additional adapter and effector proteins, generating a cascade of (CD3 zz, CD3 gε, and CD3 dε) (1). Each of these chains contains intracellular signals (6, 7). These early activation events are fol- one (CD3 g, d, ε) or three copies (CD3 z) of a conserved signaling lowed temporally by a redistribution and internalization of the domain, termed the ITAM (2, 3). Upon TCR engagement of TCR complex, which is especially important under conditions of peptide–MHC, the two tyrosine residues located within the ITAMs limiting agonist peptide–MHC concentrations on target cells (8, are phosphorylated by Src-family protein tyrosine kinases, prin- 9). All these processes are critical for T cell development, dif- cipally Lck and Fyn. In a biphosphorylated state, the ITAM is ferentiation, clonal expansion, effector functions, and/or cytokine production (6, 10). Surprisingly, the mechanistic basis for the initiation of T cell *Department of Immunology, The University of Texas Southwestern Medical Center, activation and TCR internalization/recycling remains incompletely Dallas, TX 75390; †Department of Pharmacology, The University of Texas South- understood. Experimental evidence supporting either ligand- western Medical Center, Dallas, TX 75390; ‡Department of Cell Biology, The Uni- x induced TCR clustering at the cell surface and/or conforma- versity of Texas Southwestern Medical Center, Dallas, TX 75390; and Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas, TX tional changes within the intracellular portions of the invariant 75390 chains has been presented (11–13). For example, several reports Received for publication August 11, 2010. Accepted for publication April 6, 2011. have demonstrated that TCR engagement contributes to a confor- This work was supported in part by National Institutes of Health Grants T32 mational change within the cytoplasmic tail of CD3 ε prior to AI005284 (to L.M.D.-W. and K.T.R.), AI42953, and AI71229 (to N.S.C.v.O.). ITAM phosphorylations (11–13). This conformational change Address correspondence and reprint requests to Dr. Nicolai S. C. van Oers, Depart- exposes a second signaling motif within the cytoplasmic tail of ment of Immunology, The University of Texas Southwestern Medical Center, 6000 ε Harry Hines Boulevard, Room NA2.200, Dallas, TX 75390. E-mail address: nicolai. CD3 , the proline-rich sequence, enabling it to complex one of [email protected] the Src-homology 3 domains in the Nck adapter protein. Mutation The online version of this article contains supplemental material. of the proline-rich sequence results in increased cell surface ex- + + Abbreviations used in this article: BRS, basic-rich stretch; IFT20, intraflagellar trans- pression of the TCR on developing CD4 CD8 thymocytes (14). port protein; PIP, phosphatidylinositol phosphates; PKCu, protein kinase C u; PLC-g, Further insights into the mechanistic regulation of TCR func- phospholipase C-g; PtdIns(3)P, phosphatidylinositol-3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol-5-phosphate; PtdIns tions have emerged from studies revealing that the cytoplasmic (3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol- domain of CD3 ε complexes charged phospholipids (15–17). The 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; SH2, Src- phospholipid-binding properties of CD3 ε are mediated by a basic- homology 2; WT, wild-type. rich stretch (BRS) of amino acids located just after the trans- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 membrane domain (15). Experiments using chimeric molecules www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002721 6840 CD3 z–PHOSPHOINOSITIDE COMPLEXES REGULATE T CELL ACTIVATION containing the cytoplasmic tail of CD3 ε fused to the extracellu- limpet hemocyanin-peptide immunogen using an N-terminal cysteine lar and transmembrane domain of the inhibitory NK receptor, (Pierce Chemical Co., ThermoFisher). The peptide sequence used in the KIR2DL3, revealed that interactions between CD3 ε and phos- coupling was CDTYDALHMQTLAPR. Anti–SLP-76 antisera were generously provided by Dr. Gary Korezky (University of Pennsylvania). The pholipids present on the inner leaflet of the plasma membrane anti-CD3 ε secreting hybridoma (145-2C11) was obtained from American sequester the ITAM of CD3 ε, thereby preventing its tyrosine Type Culture Collection (Manassas, VA). The anti-CD28 mAb secreting phosphorylation until receptor ligation (17). Yet, subsequent hybridoma cell line was generously provided by Dr. James Allison studies, using T cells from mice containing mutations that pre- (Memorial-Sloan Kettering, New York, NY). Anti-CD3 and anti-CD28 ε mAbs were purified from culture supernatants using standard affinity vented the BRS of CD3 subunits from complexing phosphoi- chromatography procedures. The BWd, CD3 z-deficient T cell line was nositides, revealed a more defined role for this motif in controlling kindly provided by Dr. Bernard Malissen (INSERM, Marseille, France) the cell surface expression of the TCR (15). Given that the and has been previously described (23). Biotinylated and cysteine linked phosphorylation state of the CD3 ε ITAM remained similar when peptides were synthesized by The University of Texas Southwestern comparing T cells expressing the wild-type (WT) versus BRS- Medical Center Protein Chemistry Technology Core facility. modified CD3 ε molecules, the phosphoinositide-binding proper- Constructs ties of CD3 ε likely regulate TCR downregulation and/or recy- Selected amino acid substitutions in the cytoplasmic tail of a full-length cling, rather than the initiation of TCR signaling. This is consistent cDNA of murine CD3 z were generated using overlapping oligonucleo- with the reduced TCR expression after the knockdown of the tides combined with PCR-based site-directed mutagenesis procedures, intraflagellar transport protein (IFT20), which is present in recy- based on the manufacturers’ instructions (Stratagene Products, Agilent cling endosomes and associates with CD3 ε (18, 19). Technologies, Santa Clara, CA). All nucleotide sequence changes were confirmed by dsDNA sequencing. The various lysine- and arginine- The cytoplasmic tail of the CD3 z subunit has also been reported modified constructs were subcloned into pcdef-3, pEGFP-N1, pGC, and Downloaded from to complex phospholipids (20). When a recombinant protein con- pGEX-2TK vectors. The pGEX vectors were used to generate GST–z taining just the cytoplasmic domain of CD3 z was incubated in fusion proteins according to the manufacturer’s instructions (GE Bio- the presence of liposomes, in vitro kinase reactions revealed that sciences, Piscataway, NJ). the tyrosine phosphorylation of the ITAMs was significantly in- Imaging hibited (20). Although such studies establish the ability of CD3 z The CD3 z sensors were generated by linking full-length WT CD3 z and to bind phospholipids, the region in CD3 z responsible for such http://www.jimmunol.org/ the BRS Sub-C construct to the N terminus of GFP (pGC vectors). The interactions, the phospholipid-binding specificity of CD3 z, and sensors were retrovirally transduced into primary, in vitro-primed 5C.C7 the functional role of such interactions within an intact TCR TCR transgenic T cells or D011.10 TCR transgenic T cells. The trans- have not been established. In this study, we report that CD3 z con- duced T cells were FACS sorted at defined z–GFP sensor expression levels tains a high-affinity phosphoinositide-binding sequence, localized selected to be similar to endogenous protein levels, as described previously (24). CD3 z–GFP expressing 5C.C7 or D011.10 T cells were imaged in in a BRS signaling motif preceding the second ITAM. This sequence three dimensions at various time points before and after T cell–APC selectively complexed several phosphoinositides, including phos- interactions with APCs preloaded with moth cytochrome C or OVA pep- phatidylinositol-3-phosphate [PtdIns(3)P], phosphatidylinositol 4- tides, respectively. These images were analyzed as described (15, 24, 25). phosphate [PtdIns(4)P], phosphatidylinositol-5-phosphate [PtdIns Transfections, immunoprecipitations, and T cell activation by guest on September 24, 2021 (5)P], phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], and assays phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Func- tionally, mutating the CD3 z BRS in a manner that eliminated its The transfection of HEK293 cells was performed as described (26). T cell z d phosphoinositide-binding ability significantly impaired TCR ac- lines were generated with the CD3 -deficient BW T cell hybridoma, transfected with 20 mg plasmid DNA (pcdef-3-murine CD3 z) premixed cumulation at the center of the immunological synapse. This was with the Transfast transfection reagent, according to the manufacturer’s evident with two distinct TCR transgenic T cells. Despite the instructions (Promega Corp., Madison, WI) (23). Stable clones, isolated by dramatic changes in TCR localization, proximal TCR signaling G418 drug selection, were selected by the restored expression of the TCR, pathways remained intact. Rather, the CD3 z–phosphoinositide determined by flow cytometry. For stimulations and phosphoprotein induction, the cells were incubated with biotinylated anti-CD3 ε mAbs, interactions were necessary for the normal redistribution of the followed by streptavidin cross-linking for various time points as described TCR after TCR signaling. Taken together, these findings reveal previously (27). The cells were lysed in a 1% Triton X-100–containing a previously unrecognized role for CD3 z–lipid interactions in lysis buffer, pH 7.6 (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1 mM regulating T cell functions. NaF, supplemented with protease and phosphatase inhibitors) (27). All other immunoprecipitations and immunoblotting assays were previously described (15, 28, 29). For expression of CD25 and CD69, the cells (105 Materials and Methods cells/well) were cultured in the presence of plate-bound anti-CD3 ε for Abs, peptides, and cell lines varying time points. In the case of transduced T cells, 1 3 104 T cells were incubated for 20 h with an equivalent number of CH27 B cells that had Fluorochrome-labeled Abs for flow cytometry were obtained from either been preloaded with varying peptide concentrations (0.05–6 mM). IL-2 Becton-Dickinson (Franklin Lake, NJ) or eBioscience (San Diego, CA). production was assayed with a mouse IL-2 ELISA assay according to Phospho-specific Abs against protein kinase C u (PKCu) (Thr 538; cat no. the manufacturer’s instructions (ELISA MAX mouse IL-2; BioLegend). 9377S), phospholipase C-g (PLC-g) (Ser 1248; cat. no. 4501S), ZAP-70 (Tyr 319; cat. no. 2701L), AKT (Ser473; cat. no. 9271S), and p42/p44/Erk/ Phospholipid-binding assays MAPK (Tyr 202/204; cat. no. 9101S) were obtained from Cell Signaling Technology (Danvers, MA). Anti-actin (cat. no. 4967) and anti–PLC-g Phosphatidylinositol phosphates (PIP) strips or PIP arrays were preblocked (cat. no. 2882) were also purchased from Cell Signaling. An anti–phospho- in 3% fatty acid-free BSA prepared in a Tris-based buffer (TBST; 25 mM z specific mAb (pY142; cat. no. 558402) and mAbs against Erk (cat. no. Tris, 125 mM NaCl, 0.1% Tween 20; pH 8) (Echelon Biosciences, Salt Lake 610124) and Akt (cat. no. 610861) were purchased from Becton- City, UT). The membranes were then incubated with biotinylated peptides Dickinson. A mouse mAb against SLP-76 (cat. no. 625002) was purchased or GST fusion proteins (1 and 5 mg/ml in 1% fatty acid-free BSA/TBST, from BioLegend (San Diego, CA). Anti-PKCu antisera (cat. no. sc1875) respectively) for 2 h at room temperature or overnight at 4˚C. The mem- were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). mAbs branes were subsequently washed and immunoblotted with either strep- against CD3 z (6B10.2) and ZAP-70 (IE7.2 and 2F3.2) were previously tavidin–HRP conjugates or anti-GST mAbs in combination with secondary described (21, 22). The mAb 2F3.2 recognizes the tandem SH2 domains of Abs coupled to HRP. ECL was used to detect binding (Pierce Chemical human ZAP-70. Of note, the CD3 z BRS Sub-C molecule was not rec- Co., ThermoFisher). Liposomes were prepared by sonicating the lipids in ognized by the mAb 6B10.2. Consequently, polyclonal rabbit anti-CD3 z a HEPES buffer (50 mM HEPES, pH 7, 100 mM NaCl, 1 mM EDTA) antisera (rabbit sera no. 10405 or 10407) were generated against the C- containing 0.5 M sucrose. The liposomes were then diluted in sucrose-free terminal region of murine CD3 z, which had been prepared as a keyhole buffer. GST–z WT or GST–z BRS Sub-C (4 mM) was incubated with 25 The Journal of Immunology 6841 mM phospholipid containing 800 mM brominated phosphatidylcholine for brane via lipid binding, thereby modulating its phosphorylation 10 min at 4˚C. The liposomes were pelleted by centrifugation, washed in (17). The first BRS (BRS-1) of CD3 z, located at the juxtamem- HEPES buffer, resuspended in SDS-sample buffer, and resolved by SDS- brane position, did not bind any phospholipids, unlike that PAGE. Samples were collected as supernatant or pellet, with the pellet ε containing the protein–liposome complex, as previously described (15). reported for CD3 (Fig. 1B) (15). BRS-2, encompassing a region that preceded the second ITAM, bound to almost every phosphoinositide (Fig. 1B). BRS-2 did not associate with phosphati- Results dylcholine, phosphatidylserine, phosphatidylethanolamine, or sphin- z The cytoplasmic tail of CD3 contains multiple gosine-1-phosphate, demonstrating a selective inositol binding phospholipid-binding regions specificity (Fig. 1B). BRS-3, located between the second and third The cytoplasmic domains of both CD3 ε and CD3 z can complex ITAM, preferentially bound to PtdIns(4)P, PtdIns(5)P, and phos- phospholipids, as assayed with liposomes and lipid-embedded phatidic acid, much like the binding specificity reported for CD3 ε membranes (15, 16, 20). While the CD3 ε lipid binding domain (Fig. 1B) (15). The first CD3 z (ITAM-1) complexed PtdIns(3,4,5) maps to a basic amino acid-rich stretch (BRS) that follows its P3 weakly, with some binding to PtdIns(3)P noted on longer transmembrane domain, the corresponding region(s) in CD3 z and exposures (Fig. 1B) (data not shown). Peptides encompassing the its phospholipid-binding specificity have not been established second and third ITAMs (ITAM-2 and ITAM-3) did not bind to the (15). CD3 z contains multiple clusters of basic amino acids that PIP strips. Substitution of lysine and/or arginine residues with are highly conserved in different species (Fig. 1A, Supplemental alanines, valines, and glycines in ITAM-1 (ITAM-1 Sub), BRS-2 Fig. 1). To define which, if any, of these clusters were capable of (BRS-2 Sub), and BRS-3 (BRS-3 Sub) completely eliminated mediating phospholipid interactions, biotinylated peptides span- phospholipid binding, confirming the importance of the positively Downloaded from ning distinct subsections of CD3 z were used to probe membranes charged amino acids in mediating the CD3 z–phospholipid inter- embedded with an assortment of lipids (PIP strips). The peptides actions (Supplemental Fig. 2A,2B). included three polybasic clusters, termed BRS-1, BRS-2, and BRS- To better define the lipid-binding affinities of ITAM-1, BRS-2, 3, as well as the three ITAMs (ITAM-1 to -3) (Fig. 1A). The indi- and BRS-3, the corresponding peptides were used to probe PIP vidual ITAMs were tested because the CD3 ε ITAM has been arrays containing serial dilutions of the phosphatidylinositol

proposed to embed within the inner leaﬂet of the plasma mem- phospholipids (Fig. 1C). BRS-2 had the highest relative afﬁnity for http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. The cytoplasmic tail of CD3 z contains multiple clusters of arginine and lysine residues that complex phosphoinositides. A, Amino acid sequence of the cytoplasmic tail of murine CD3 z containing clusters of basic amino acids, as indicated by asterisks. Individual biotinylated peptides containing the three BRS sequences (BRS-1, BRS-2, BRS-3) and the three ITAMs (ITAM-1, ITAM-2, ITAM-3) are shown. The tyrosine residues present in the ITAMs are underlined. B, PIP strips embedded with diverse lipids were probed with the biotinylated peptides. Binding was detected by streptavidin– HRP Western blotting, with the different exposure times indicated below the membrane. Data are representative of four independent experiments. C, Biotinylated peptides containing ITAM-1, BRS-2, and BRS-3 were used to immunoblot lipid arrays containing serial dilutions of the indicated phospholipids ranging from 100 to 1.56 pmol, as shown in the left panel. Peptide binding was assessed as in B. The data are representation of at least four independent binding assays per peptide. 6842 CD3 z–PHOSPHOINOSITIDE COMPLEXES REGULATE T CELL ACTIVATION

PtdIns(5)P, followed by PtdIns(3,5)P2, binding amounts as low as Sub-C). These modifications comprised 12, 9, and 4 aa sub- 3.1 pmol (Fig. 1C). BRS-3 required at least 25 pmol of PtdIns(5)P, stitutions, respectively (Fig. 2A). The GST–BRS Sub-A, -B, and PtdIns(3,5)P2, and PtdIns(4)P, suggesting a binding affinity at least -C fusion proteins did not bind any phosphoinositides (Fig. 2B). 30-fold less than BRS-2. ITAM-1 complexed to PtdIns(3,4,5)P3, The fact that substitution of only four residues (BRS Sub-C) but required a minimum of 25 pmol of spotted lipid for detection. eliminated lipid associations suggests that the positively charged These binding affinities differed from each other as well as those sequence located within the second BRS (BRS-2) is the principal observed with the CD3 ε BRS (Supplemental Fig. 2C). phospholipid-binding motif in the cytoplasmic tail of CD3 z. This was supported by the 30-fold higher affinity of the BRS-2 peptide z A single BRS within the cytoplasmic tail of CD3 is the for phosphoinositides compared with the other phospholipid- predominant phospholipid-binding motif binding peptides (ITAM-1 and BRS-3). We next examined We next examined the phospholipid-binding specificity of the whether CD3 z could complex phospholipids present in a lipid entire cytoplasmic tail of CD3 z using purified GST fusion proteins bilayer. Liposomes containing 80% phosphatidylcholine mixed (Fig. 2A). The full cytoplasmic region of CD3 z had an affinity with 20% of either phosphatidylinositol-4,5-bisphosphate [PtdIns similar to the BRS-2 peptide (Fig. 2B). To determine the contri- (4,5)P2] or PtdIns(5)P were incubated with GST, GST–z (BRS bution of the basic amino acid clusters in this binding, several WT), or GST–BRS Sub-C (Fig. 2C). Greater than 90% of the CD3 z-modified constructs were generated in which alanine, control GST protein was retained in the supernatant, with little glycine, and valine residues were substituted for the indicated detected in the liposome-containing pellet (Fig. 2C, lanes 1 and 3 arginines and lysines. Because lysine residues in CD3 z can serve versus 2 and 4). In contrast, 78% of GST–z (GST–z WT) was as ubiquitination sites, the majority of the mutations were in- associated with PtdIns(4,5)P2, and ∼37% complexed with PtdIns Downloaded from troduced at the arginine positions (30). The substitutions were (5)P-containing liposomes (Fig. 2C, lanes 6 versus 5 and 8 versus introduced in all three binding subregions (BRS Sub-A), within 7). GST–BRS Sub-C exhibited much less lipid binding to PtdIns ITAM-1 and BRS-2 (BRS Sub-B), or just within BRS-2 (BRS (4,5)P2 (26%), with the majority (74%) detected in the supernatant http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. A single polybasic cluster in the cytoplasmic tail of CD3 z complexes selected phosphoinositides with high afﬁnity. A, Amino acid sequence of the cytoplasmic domain of WT CD3 z and constructs containing a number of amino acid substitutions at various polybasic cluster positions. These include substitutions of 12 (BRS Sub-A), 9 (BRS Sub-B), and 4 (BRS Sub-C) residues, respectively. The substitutions are boldface and underlined. B, PIP arrays containing serial dilutions of the indicated phosphoinositides were probed with the puriﬁed GST–z fusion proteins. Binding was detected by anti- GST Western blotting assays. Binding assays with the substituted constructs are shown with 2-min (BRS Wild Type) and 30-min (BRS Sub-A, -B, -C) exposures. The data are representation of three independent experiments. C, Sucrose-loaded liposomes, consisting of the indicated phospholipids, were incubated with GST (lanes 1–4), GST–z Wild Type (lanes 5–8), or GST–BRS Sub-C BRS (lanes 9–12). Proteins retained in the supernatant (S) or pellet (P, liposome binding fraction) were detected by SDS-PAGE analysis using Coomassie brilliant blue staining. The percentage of GST fusion protein detected in the supernatant and pellet is listed under the appropriate lanes. These results were obtained in two independent experiments. The Journal of Immunology 6843

(lanes 9 and 10). There was ∼40% GST–BRS Sub-C detected in phosphorylated CD3 z intermediates (27). Accordingly, each of the pellet in the presence of PtdIns(5)P (Fig. 2C, lanes 10 and 12). the CD3 z BRS Sub molecules was more heavily phosphorylated The fact that some BRS Sub-C was detected in the pellets con- when coexpressed with ZAP-70 (Fig. 3D,3E). This was consistent taining either PtdIns(4,5)P2 and PtdIns(5)P was likely caused by with the increased percentage of CD3 z that was tyrosine phos- the propensity of this construct to precipitate in solution (data not phorylated (Fig. 3F versus 3C). To ascertain whether mutations in shown). Taken together with the lipid arrays, our findings dem- CD3 z affected ZAP-70 association, the CD3 z precipitates were onstrate that CD3 z contains a phosphoinositide binding domain. immunoblotted with anti–ZAP-70 Abs. ZAP-70 complexed the various phospho-CD3 z intermediates, with the largest amount of The phospholipid-binding properties of CD3 z are uncoupled ZAP-70 coprecipitating with BRS Sub-C (Fig. 3G). Taken to- from the early TCR-induced phosphoprotein induction gether, these experiments demonstrated that the CD3 z BRS The lipid-binding properties of the cytoplasmic domains of both interactions with phosphoinositides are not required for ITAM CD3 ε and z have been previously reported to regulate the tyrosine accessibility to Src-family kinases or phospho-ITAM binding to phosphorylation of their ITAMs (17, 20). Furthermore, phos- ZAP-70. phorylation of the CD3 z ITAMs was found to prevent the lipid- To examine further the functional role of the CD3 z BRS– binding activity of its cytoplasmic tail (20). In accordance with phosphoinositide interactions in regulating signaling pathways, these studies, we found that GST–z constructs containing phos- CD3 z-deficient T cells were reconstituted with WT or BRS Sub-C phorylated ITAMs exhibited a significant reduction in their ability CD3 z chains. Clones were selected for identical TCR surface to bind numerous phospholipids present on the PIP arrays (Sup- expression and compared for their capacity to initiate TCR- plemental Fig. 3). Together, these data suggest that modifications mediated phosphoprotein induction at different time points. The Downloaded from to the phospholipid-binding properties of full-length CD3 z could magnitude and kinetics of overall TCR-mediated phosphoprotein impact ITAM phosphorylations and/or ZAP-70 associations. To induction was comparable in the WT and BRS Sub-C recon- address these possibilities, we characterized the phosphorylation stituted T cells (Fig. 4A). These findings were consistent with that potential of the CD3 z constructs containing the various BRS observed in HEK cells, suggesting that the phosphoinositide- modifications. Experimentally, HEK cells were transfected with binding functions of CD3 z are unconnected from early in-

constructs containing Lck and/or ZAP-70 (tandem SH2 domain) tracellular signaling events. To address this question more care- http://www.jimmunol.org/ in addition to either vector alone or the indicated CD3 z constructs fully, selected proteins were either immunoprecipitated followed (WT, BRS Sub-A, Sub-B, or Sub-C) (Figs. 2A,3A and 3B). In the by Western blotting or directly assayed with anti–phospho-specific absence of ZAP-70, WT CD3 z appeared as a heterogeneous set of Abs. These experiments revealed similar levels and kinetics of phosphoproteins ranging from 18 to 23 kDa when analyzed in ZAP-70 (Fig. 4B), CD3 z that co-precipitated with ZAP-70 (Fig. total cell lysates (Fig. 3A, lane 3). The BRS Sub-A and -B also 4B), PLC-g (Fig. 4D), PKCu (Fig. 4E), AKT (Fig. 4F), and Erk developed into several phosphorylated intermediates, although (Fig. 4G) phosphorylation in WT and BRS Sub-C expressing both exhibited much higher molecular masses of 26–30 kDa (Fig. T cells (Fig. 4B–G, Supplemental Fig. 4). The only protein that 3A, lanes 4 and 5). The CD3 z BRS Sub-C protein was tyrosine showed a statistically significant difference between the two phosphorylated and migrated in a pattern very similar to WT CD3 clones was SLP-76 (Fig. 4C, t = 3 min, p = 0.047) (Supplemental by guest on September 24, 2021 z, although with a slightly faster mobility (Fig. 3A, lane 6 versus Fig. 4). Importantly, both clones expressed similar levels of total 3). The phosphorylation patterns were confirmed by direct im- actin whereas the levels of CD3 z were slightly lower in the BRS munoprecipitation of CD3 z followed by anti-phosphotyrosine and Sub-C line (Fig. 4H,4I). TCR signaling processes are required for anti-CD3 z immunoblotting (Fig. 3B,3C). These experiments the expression of activation markers, including CD69 and CD25. demonstrated that elimination of the phosphoinositide-binding To determine if the phosphoinositide-binding function of CD3 z functions of CD3 z did not affect its tyrosine phosphorylation in contributes to these later processes, TCR-induced upregulation of HEK cells. This contrasts the increased phosphorylation noted for CD69 and IL-2 secretion was compared between the WT and BRS CD3 ε when its BRS was mutated, an effect only found in non- Sub-C reconstituted T cells. There was no significant impairment T cells (15). in CD69 induction or IL-2 production in T cells lacking the lipid- Coexpression of the tandem SH2 domains of ZAP-70 with CD3 binding functions of CD3 z (data not shown). These results z results in the binding and stabilization of the more heavily demonstrated that the phosphoinositide-binding functions of CD3

FIGURE 3. The tyrosine phosphorylation of the CD3 z ITAMs is uncoupled from the lipid-binding functions of z. A, HEK293 cells were cotransfected with Lck and control vector (lane 1) or the various CD3 z constructs including WT CD3 z (lane 2), BRS Sub-A (lane 3), BRS Sub-B (lane 4), and/or BRS Sub- C(lane 5). The cells were processed for immunoblotting with anti-phosphotyrosine. B and C, CD3 z was immunoprecipitated from the lysates prepared in A and immunoblotted with anti-phosphotyrosine (B) and anti-CD3 z mAbs (C). D–F, The cells were transfected as in A along with an expression vector containing the tandem SH2 domains of ZAP-70. D, Samples were processed for immunoblotting of total cell lysates with anti-phosphotyrosine mAbs. E–G, CD3 z was immunoprecipitated from the cell lysate and blotted with mAbs against phosphotyrosine (E), CD3 z (F), and ZAP-70 (G). The results were con- ﬁrmed in ﬁve independent experiments. 6844 CD3 z–PHOSPHOINOSITIDE COMPLEXES REGULATE T CELL ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 4. TCR-mediated activation of early TCR signaling processes is independent of the CD3 z–lipid binding interactions. CD3 z-deficient T cells (BWd) were reconstituted with WT z or the BRS Sub-C construct. Clones, selected for equivalent TCR expression, were left untreated or stimulated with anti-CD3 mAbs for the indicated times (0, 3, 10, and 30 min). Total cell lysates (A) or immunoprecipitates of ZAP-70 (B) and SLP-76 (C) were prepared for immunoblotting with anti-phosphotyrosine. In addition, anti–phospho-specific antisera and Abs against a number of additional proteins were used to by guest on September 24, 2021 immunoblot total cell lysates. These included anti–phospho-PLC-g and anti–PLC-g (D), anti–phospho-PKCu (E), anti–phospho-AKT and anti-AKT (F), anti–phospho-Erk and anti-Erk (G), and anti-actin (H) and anti-CD3 z (I). Data are representative of three independent experiments. z are not required for early TCR-mediated phosphoprotein in- instead accumulating in internal punctuate structures, most likely duction after a strong stimulus. vesicles (Fig. 5A,5C, Supplemental Video 1). Such internal accumulation was seen in 88 6 4% of the cell couples expressing BRS The phospholipid-binding properties of CD3 z are required for Sub-C–GFP, and less than half with cell couples with WT CD3 z– normal immunological synapse formation GFP (p , 0.001). In addition, the percentage of cells exhibiting any T cell interactions with agonist peptide-loaded APCs causes the TCR accumulation of CD3 z at the interface after 7 min of tight cell to redistribute to a structure at the center of the T cell–APC interface couples formation dropped significantly from 93 6 5% in T cells known as the central supramolecular activation cluster or immu- expressing WT z–GFP to 24 6 5% in T cells expressing BRS Sub- nological synapse (31). Eliminating the positively charged residues C–GFP (p , 0.001). The experiments were repeated with a distinct within the BRS of CD3 ε was found to impair significantly the TCR transgenic line, D011.10, which expresses an OVA-specific recruitment of the TCR to the immunological synapse (15). To TCR. Consistent with the 5C.C7 results, the TCR in the BRS examine whether the CD3 z binding to phosphoinositides could Sub-C–GFP expressing D011.10 T cells was not maintained at the have a similar function, 5C.C7 T cells were reconstituted with the T cell–APC interface for more than about 2 min and again accu- various CD3 z–GFP constructs, and imaging studies were per- mulated in internal punctuate structures (Fig. 5E versus 5D, Sup- formed to monitor the distribution of CD3 z. Quantitative immu- plemental Video 2). This internal accumulation was seen in 88 6 noblotting of the GFP-sorted cells indicated that the levels of the 5% of the cell couples, very distinct from the WT CD3 z–GFP (p , z–GFP chimeric molecule were 14.4 6 7% (n = 3) overexpressed 0.001) (Fig. 5D). Unlike the 5C.C7 T cells, the accumulation of the compared with the endogenous protein levels (Supplemental Fig. 5). TCR at the distal pole in the D011.01 cells was not affected by the Upon tight cell couple formation, the localization of BRS Sub-C to mutation of the BRS. The localization defects noted with CD3 z two prototypical sites of TCR accumulation (the distal pole and the were distinct from those observed when analyzing the redistribution center of the T cell–APC interface) was dramatically impaired (Fig. of other CD3 chains containing various signaling mutations. For 5A). The number of cell couples exhibiting transient accumulation example, mutation of the CD3 ε BRS motif resulted in delayed and of CD3 z–GFP at the distal pole of the T cell was significantly reduced accumulation of the TCR complex at the center of the reduced (p , 0.001) from a maximum of 64 6 7% to 20 6 4% T cell–APC interface but without the consistent and prominent in- when comparing WT CD3 z–GFP with BRS Sub-C–GFP (Fig. 5B ternal accumulation seen with the CD3 z BRS Sub-C–GFP con- versus 5C). Unlike WT CD3 z, BRS Sub-C–GFP was not main- structs (15). Moreover, substitution of the tyrosine residues in the tained at the center of the T cell–APC interface for more than 120 s, CD3 z ITAMs yielded a slower translocation of CD3 z from the The Journal of Immunology 6845 Downloaded from http://www.jimmunol.org/

FIGURE 5. The spatiotemporal accumulation of CD3 z at the immunological synapse is partially regulated by its phospholipid-binding properties. 5C.C7 (A–C) or D011.10 (D, E) TCR transgenic T cells were retrovirally transduced with constructs encoding the WT CD3 z or BRS Sub-C, both linked to GFP. The GFP-positive cells were imaged after incubations with 10 mM peptide loaded APCs (moth cytochrome C). A, Representative interaction of CD3 z Sub- C–GFP 5C.C7 T cells with the APC is shown at the indicated time points (seconds) relative to the time of formation of a tight cell couple. Differential by guest on September 24, 2021 interference contrast images are shown in the top row (GFP, original magnification 340), with top-down, maximum projections of three-dimensional z Sub- C–GFP fluorescence data in the bottom row. The fluorescence intensity is displayed in a rainbow-like false-color scale (increasing from blue to red). A video covering the entire time frame is available in Supplemental Video 1. B and C, The graphs display the percentage of cell couples from experiments similar to those of A with accumulation of WT CD3 z–GFP (B) and Sub-C–GFP (C). Any accumulation refers to z–GFP molecules that are grouped at the entire interface formed between the T cell and APC. Central accumulation refers to the clustering of z–GFP at the very center of the T cell–APC interface. Peripheral accumulation is for the clustering of z–GFP molecules at the outside ring of the T cell–APC interface. Finally, distal accumulation is evident when there is a clustering of z–GFP at the distal pole of the T cell, opposite to the area of T cell–APC contact. These patterns are described in detail elsewhere (15, 24). D and E, DO11.10 T cells were mixed with A20 B cell lymphoma APCs in the presence of 10 mM OVA peptide. The graphs display the percentage of cell couples with accumulation of WT z–GFP (D) or BRS Sub-C–GFP (E) as described in A–C. A total of 45, 90, 85, and 50 cell couples were analyzed in B, C, D, and E, respectively. Differences in accumulation in any interface pattern between WT and BRS Sub-C were significant (p , 0.001) at all time points $100 s; differences in central accumulation were significant (p , 0.005) at all time points $40 s. A video covering the entire time frame for the OTII cells is available in Supplemental Video 2. distal pole to the center of the T cell–APC interface, without af- cytosolic proteins to bind lipids. In the immune system, the cy- fecting interface accumulation (24). These data support a unique toplasmic domains of several ITAM-containing subunits, in- role for the CD3 z BRS signaling motif in the redistribution of the cluding CD3 ε and FcεRIg, have been shown to bind phos- TCR after T cell–APC interactions. pholipids due to the presence of such polybasic structures (15, 16, Using these retrovirally reconstituted T cells, we next analyzed 20). In this study, we report that the CD3 z subunit contains late T cell activation events. In a peptide dose-response curve, the a key polybasic cluster—which precedes the second ITAM— levels of IL-2 and the induction of CD69 expression were that complexes select phosphoinositides including PtdIns(3)P, equivalent between the WT and CD3 z BRS Sub-C reconstituted PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 with a cells (data not shown). These findings suggest that while CD3 z– high affinity. CD3 z does not bind phosphatidylethanolamine, phos- phospholipid interactions stabilize synapse formation, this is not phatidylserine, phosphatidylcholine, or sphingosine-1-phosphate. critical for induction of CD69 expression and IL-2 production. Because the structural basis of polybasic cluster–phosphoino- This could relate to the presence of endogenous CD3 z molecules sitide interactions is not well understood, we engineered diverse in the 5C.C7 cells and the fact that the TCR contains two in- arginine and lysine substitutions in CD3 z to eliminate any basic dependent signaling modules, CD3 zz and CD3 gεdε (32). amino acid pairs. As lysine residues are polyubiquitination sites, the substitutions were directed at three arginine residues and just Discussion one lysine (30, 33). The one lysine substituted (in murine CD3 z Polybasic clusters are defined as arginine- and lysine-enriched BRS Sub-C) does not undergo K-33 linked polyubiquitination amino acid sequences that enable diverse transmembrane and and is not conserved in other species (34). Based on the various 6846 CD3 z–PHOSPHOINOSITIDE COMPLEXES REGULATE T CELL ACTIVATION amino acid substitutions that were engineered in CD3 z, it appears to be essential for the initiation of TCR signal transmission (17, that the phosphoinositide binding requires a tandem pair of basic 35). Although CD3 z and ε might contribute to this process, the residues separated by 10 other amino acids. lipid-binding functions are clearly not required for TCR signaling In prior studies, using just the cytoplasmic region of CD3 z, the after TCR interactions with cognate peptide–MHC complexes. As ability of Src-kinases to phosphorylate the tyrosine residues within our data establish, CD3 z–phosphoinositide interactions regulate its ITAMs was inhibited in the presence of liposomes containing TCR clustering, internalization, and/or recycling. The combined negatively charged phospholipids (20). These observations led to ability of CD3 z to bind to diverse phosphoinositides is likely to be the hypothesis that electrostatic interactions between the cyto- essential for T cell activation under situations of low peptide– plasmic tail of CD3 z and charged phospholipids may regulate the MHC concentrations. In addition to CD3 z and ε, the FcεRI phosphorylation state of its ITAM (35). A similar mechanism has g-chain has also been reported to complex lipids. Our findings been proposed for CD3 ε, where the phosphorylation of its ITAM suggest that this lipid-binding property could also influence the was hypothesized to be blocked by insertion of the cytoplasmic clustering and aggregation of the high-affinity IgE receptor (16, tail into the plasma membrane (17, 35). However, all these con- 41). Other proteins of the innate and adaptive immune system that clusions were based on experiments using recombinant proteins contain BRS motifs include stromal interaction molecule 1, a lacking the extracellular and transmembrane regions of the CD3 calcium regulator, and Trif-related adapter molecule, a TLR adapter molecules in the absence of an intact TCR complex. To define protein (42, 43). Ultimately, understanding the role of CD3 z– the functional contribution of CD3 z–phosphoinositide binding in phospholipid interactions in T cell functions will provide greater T cells, we expressed a GFP-linked CD3 z BRS Sub-C molecule insight into not only the mechanisms controlling T cell activation in naive 5C.C7 and OTII T cells. Remarkably, a functional phos- but also how other proteins containing such polybasic clusters Downloaded from pholipid interaction was required for effective recruitment of the regulate cellular activity. TCR to the immunological synapse. Moreover, the inability to bind phosphoinositides resulted in an intracellular accumulation Acknowledgments of CD3 z in punctuate structures. This suggests that efficient re- We thank Angela Mobley for assistance with flow cytometry. We appreciate distribution of CD3 z after Ag receptor signaling requires CD3 z the efforts of Sabrina Martinez-Anz and Sarah Ireland in analyzing phos- to complex select phosphoinositides. Although the identity of the pho-z and CD3 z BRS Sub binding to the lipid arrays. All the peptides http://www.jimmunol.org/ punctuate structure is unknown, recent experiments suggest that it were synthesized by the Chemistry Core facility at The University of Texas could be recycling endosomes. Imaging studies have revealed an Southwestern Medical Center. accumulation of phospho-z in endosomes (36). In addition, recycling endosomes containing TCR–CD3 polarize to the immuno- Disclosures logical synapse (18). These distributions could require that CD3 z The authors have no financial conflicts of interest. BRS interacts with PtdIns3P, PtdIns(3,5)P2 and low levels of PtdIns(3,4,5)P3, which are enriched in endosomes and the plasma membrane, respectively. A small amount of PtdIns(4)P is also References generated at the plasma membrane after T cell activation (19, 37– 1. Call, M. E., J. Pyrdol, and K. W. Wucherpfennig. 2004. Stoichiometry of the T- by guest on September 24, 2021 40). 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