A large Rab GTPase encoded by CRACR2A is a component of ...stke.sciencemag.org/content/sigtrans/9/420/ra31.full.pdf · A large Rab GTPase encoded by CRACR2A is a component of subsynaptic

R E S E A R C H A R T I C L E

I M M U N O L O G Y

A large Rab GTPase encoded by CRACR2A is acomponent of subsynaptic vesicles that transmitT cell activation signalsSonal Srikanth,1*† Kyun-Do Kim,1†‡ Yuanyuan Gao,1§ Jin Seok Woo,1 Shubhamoy Ghosh,1¶

Guillaume Calmettes,2 Aviv Paz,1 Jeff Abramson,1 Meisheng Jiang,3 Yousang Gwack1*

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More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the humangenome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, andfusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodesa lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand mo-tifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site.Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role forCRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response toT cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi tothe immunological synapse formed between a T cell and a cognate antigen-presenting cell to activatethese signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotideexchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse.Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with itslocalization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of alarge Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain ar-chitectures may have similar functions in T cells.

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INTRODUCTION

The antigen-dependent activation of T cells requires their direct contactwithantigen-presenting cells (APCs). The binding of T cell receptors (TCRs) tocognate peptide-bound major histocompatibility complexes on APCs in-duces clustering of the TCRs and recruitment of lymphocyte-specific pro-tein tyrosine kinase (Lck) and z-chain–associated protein kinase of 70 kD(ZAP70). These kinases phosphorylate the signaling adaptor protein Lat,which forms a signalosome containing phospholipase C-g1 (PLC-g1) andthe guanine nucleotide exchange factor (GEF) and adaptor molecule Vav1(1–3). The activity of PLC-g1 produces the second messenger inositol1,4,5-trisphosphate (IP3), which binds to the IP3 receptor on the endoplas-mic reticulum (ER) and stimulates the depletion of the ER Ca2+ store. Bysensing the depletion of Ca2+ from the ER, stromal interaction molecule1 (STIM1) translocates to the plasma membrane–proximal regions of theER and activates Orai1, the pore subunit of the Ca2+ release–activatedCa2+ (CRAC) channels (4–6). Vav1 accumulates at the immunological syn-apse and recruits small G proteins (guanine nucleotide–binding proteins),such as Rac1 and CDC42 (cell division control protein 42 homolog), to ac-

1Department of Physiology, David Geffen School of Medicine, University of Cali-fornia, Los Angeles (UCLA), Los Angeles, CA 90095, USA. 2Department of Med-icine (Cardiology), David Geffen School of Medicine, UCLA, Los Angeles, CA90095, USA. 3Department of Molecular and Medical Pharmacology, David Gef-fen School of Medicine, UCLA, Los Angeles, CA 90095, USA.*Corresponding author. E-mail: [email protected] (Y.G.); [email protected] (S.S.)†These authors contributed equally to this work.‡Present address: University of Rochester Medical Center, Rochester, NY14642, USA.§Present address: Laboratory of Immunogenetics, Virology and Cellular Im-munology, National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health, Rockville, MD 20852, USA.¶Present address: Department of Molecular, Cell, and Developmental Biolo-gy, College of Life Sciences, UCLA, Los Angeles, CA 90095, USA.

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tivate the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated proteinkinase (MAPK) pathways (7). Activation of both the Ca2+ and MAPKsignaling pathways is essential for the differentiation of naïve T cells intoThelper cells, anddysregulationof these pathways results invarious immune-related diseases in humans and mice (8–10).

In addition to its localization at the plasma membrane, Lat also exists insubsynaptic vesicles that translocate to the plasma membrane–proximal re-gions of the immunological synapse after TCR stimulation (11–13). Re-cruitment of this pool of Lat may be important for its phosphorylation.Lat-containing vesicles use a soluble N-ethylmaleimide–sensitive factor at-tachment protein receptor (SNARE)–dependent trafficking mechanism fortheir recruitment. The v-SNARE protein VAMP7 guides these vesicles tothe plasma membrane potentially by docking to t-SNARE proteins througha mechanism that does not involve membrane fusion (14). These resultssuggest that components or functional homologs of the molecular machin-ery used for the trafficking of synaptic vesicles in the neuronal synapse,such as SNAREs and small Rab guanosine triphosphatases (GTPases), playan important role in the trafficking of subsynaptic vesicles in T cells. How-ever, the importance of these subsynaptic vesicles in TCR signaling hasbeen uncovered only recently, and the identities and functions of these sub-synaptic vesicles in T cell activation need further investigation.

More than 60 Rab GTPases exist in humans to regulate vesiculartrafficking between organelles. Rab GTPases broadly control vesiclebudding, uncoating, motility, and fusion through the recruitment of effectormolecules, including sorting adaptors, tethering factors, and motors(15, 16). The functions of Rab GTPases, such as membrane association,are regulated by both GTP binding and prenylation (the attachment of iso-prenoid lipids) (17). GTP-bound Rab GTPases are retained at the donormembrane to initiate trafficking to the target organelles, whereas guanosinediphosphate (GDP)–bound forms (which are formed after GTP hydrolysis)detach from the membrane and move to the cytoplasm. GDP-bound RabGTPases are recycled by the exchange of GDP for GTP. The C-terminal

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prenylation of RabGTPases is also essential for their associationwith donoror target membranes. The types of isoprenoid units that are attached dependon the different GTPase families. Ras GTPases are farnesylated by farnesyltransferase, whereas Rac andRhoGTPases are geranylgeranylated by type Igeranylgeranyl transferase (GGT). Rab GTPases are also geranylgerany-lated; however, this is performed by type II GGT. Statins are useful toolsto investigate protein prenylation because they are inhibitors of 3-hydroxyl-3-methyl-glutaryl–coenzymeA (HMG-CoA) reductase, the key, rate-limingenzyme in the cholesterol synthesis pathway. Statins suppress the generationof farnesyl and geranygeranyl pyrophosphate,which are substrates of prenyltransferases, and thus, these drugs inhibit the prenylation of smallG proteins(18). Statins, including atorvastatin, arewidely prescribed for their cholesterol-lowering effects. Statins also inhibit TCR signaling, and these drugs areused in clinics to suppress autoimmune diseases (19–21). These results em-phasize the critical role of the prenylation of small G proteins in intracellularsignaling for T cell activation.

Although the role of Rab GTPases in membrane trafficking is wellstudied, little is known about their direct involvement in intracellularsignaling. Moreover, our current understanding of the Rab GTPase familyis mostly limited to roles of small proteins of 20 to 25 kD in size. Here, wereport a previously uncharacterized function of a distinct large RabGTPase,CRACchannel regulator 2A (CRACR2A) isoform a (CRACR2A-a),whichcontains multiple functional domains, including the previously identifiedN-terminal CRAC channel–regulating domain (22), a proline-rich domain(PRD) for protein-protein interactions, and a C-terminal Rab GTPase do-main. Our results showed that various domains of this large Rab GTPasecontributed to its translocation from the Golgi-proximal area to the immu-nological synapse in subsynaptic vesicles, which were distinct from thoseknown to contain LAT, to activate the Ca2+-dependent transcription factor nu-clear factor of activation T cells (NFAT) and the JNK signaling pathway.Mechanistically, the PRD of CRACR2A-a was important for the recruit-ment of these vesicles to the immunological synapse through an interactionwith Vav1, whereas the GTPase domain regulated the membrane associa-tion of CRACR2A-a by GTP binding and prenylation. These observationsprovide insights into the role of subsynaptic vesicles in mediating thesignaling pathways required for T cell activation.

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RESULTS

A GTP-GDP switch regulates the association of the largeRab GTPase CRACR2A-a with the GolgiThe human genome database shows two transcriptional isoforms ofCRACR2A: CRACR2A isoform a (NM_001144958.1, CRACR2A-a)and isoform c (NM_032680.3, CRACR2A-c); however, the currentmouse genome database annotates only one isoform: CRACR2A-c(NM_001033464.3). In a previous study, we showed that CRACR2A-ccontains N-terminal EF hands and two coiled-coil domains that are requiredfor CRAC channel function (22). The other isoform, CRACR2A-a, is ofparticular interest to us because, on the basis of its predicted amino acidsequence, it contains the Orai1 and STIM1-interacting domain (which areshared by CRACR2A-c) together with other potential functional domains(Fig. 1A). First, we validated the presence of CRACR2A proteins in Jurkatcells, a human CD4+ T cell leukemia cell line, byWestern blotting analysis(CRACR2A-a, ~90 kD; CRACR2A-c, ~45 kD) (fig. S1A). Next, to con-firm the conserved function of the Orai1 and STIM1-interacting domain inCRACR2A-a, we reconstituted its expression in Jurkat cells depleted ofboth CRACR2A isoforms. Consistent with previous observations, CRACR2Adepletion resulted in a profound reduction in store-operated calcium entry(SOCE) (fig. S1, B andC). Exogenous expression of either CRACR2A-a or

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CRACR2A-c rescued SOCE in CRACR2A-depleted Jurkat cells, validat-ing their conserved function in CRAC channel activation.

Compared to CRACR2A-c, CRACR2A-a contains an additional PRDand a Rab GTPase domain that contains a predicted prenylation site in its Cterminus (Fig. 1A). The amino acid sequence of the GTPase domain ofCRACR2A-a is similar to that of the Rab GTPases, and three-dimensionalhomology modeling of this domain to a high-resolution crystal structure ofRab3a (23) showed almost complete overlap (Fig. 1B). The predictedGTPase domain also closely resembles that of other small G proteins, in-cluding Rac1 and CDC42, in the overall core fold (fig. S2) (23). TheGTPase domain contains the characteristic P-loop, as well as the switch Iand switch II regions of small G proteins. The P-loop and the switch I regionmake contact with the g-phosphate of GTP throughMg2+ ions, whereas theswitch II region plays a major role in the hydrolysis of GTP through its in-teraction with phosphates and water molecules (23, 24). To confirm theroles of these conserved residues, we generated a mutant CRACR2A-a thatinvolved substitution of an asparagine (N) residue for a threonine (T) residuewithin the P-loop, which is predicted to cause preferential GDP binding(T559N) (16, 24). The glutamine residue (Q) in the switch II region, whichserves as a catalytic residue for intrinsic GTP hydrolysis, was replaced witha leucine residue (Q604L) (24–26). The Q604L mutant CRACR2A-a pro-teinwas predicted to be in a constitutivelyGTP-bound form. The conserved,predicted guanidine-binding G4 motif of CRACR2A-a contained an aspar-agine residue, which was replaced with an isoleucine to generate the N658Imutant. Thismutationwas predicted to abolish the binding of bothGTP andGDP. Wild-type CRACR2A-a hydrolyzed GTP, which validated the pres-ence of a functional GTPase domain in its C terminus (Fig. 1C). TheT559N, Q604L, and N658I mutant CRACR2A-a proteins exhibited re-duced GTPase activity, possibly due to defects in GTP binding (T559Nand N658I) or GTP hydrolysis (Q604L). These results suggest that theGTPase domain of CRACR2A-a, which is similar to those of small G pro-teins, is functionally active.

Rab GTPases are predominantly involved in vesicle trafficking and of-ten localize in theGolgi and in secretory vesicles. Therefore, we determinedthe localization of fluorescently tagged CRACR2A-a by costaining Jurkatcells for various Golgi markers, including GM130, Golgin97, and Rab8a.CRACR2A showed the greatest colocalization with Rab8a, which marksthe trans-Golgi network (TGN) and associating vesicles (fig. S3A) (27).Endogenous CRACR2A-a also showed substantial colocalization withRab8a (fig. S3B). Therefore, we concluded that CRACR2A-a was loca-lized primarily at the Golgi-proximal area, and, more specifically, withthe membranes of the TGN and associating vesicles in resting T cells.CRACR2A-awas distinct compared to the small GTPases that we testedbecause of its predominant localization to the Golgi membrane, whereasothers, such as Rac1 and CDC42, showed a broad distribution betweenthe Golgi membrane and the cytoplasm (fig. S3C). Next, we examinedhow the binding of GDP or GTP influenced the intracellular localizationof CRACR2A-a. The GDP-bound T559N mutant and the guanidinebinding–defective mutant N658I showed a predominantly cytoplasmic lo-calization, whereas the GTP-bound Q604L mutant localized to the Golgi,similar towild-typeCRACR2A-a (Fig. 1D and fig. S4). Together, these datasuggest that CRACR2A-a localizes to theGolgimembrane in restingT cellsand that GTP binding regulates its localization.

CRACR2A-a plays a role in the activation of the JNKsignaling pathway in response to TCR stimulationTo determine whether CRACR2A-a was important for intracellularsignaling pathways other than Ca2+ signaling, we examined the extent ofphosphorylation of various signaling molecules in control and CRACR2A-deficient T cells upon TCR stimulation.Whereas CRACR2A depletion did




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Fig. 1. GTPbinding regulates the localization ofCRACR2A-aat theGolgi andthe activation of JNK by CRACR2A-a after TCR stimulation. (A) Schematicshowing the predicted domain structure of human CRACR2A-a. BothCRACR2A-a and CRACR2A-c share EF-hand motifs, coiled-coil domains(CC1 and CC2), and a leucine-rich region (LR), which interact with theOrai1-STIM1 complex to regulate Ca2+ entry. In addition, CRACR2A-acontains a PRD and a predicted Rab GTPase domain with a putative pre-nylation site at the C terminus. The fragments andmutants of CRACR2A-aused in this study are indicated. (B) Left: Homology modeling of theGTPase domain of CRACR2A-a (yellow) with Rab3a (red). The GTPasedomains of CRACR2A-a and Rab3a share 46% sequence identity and65% sequence similarity (Clustal Omega). Right: MODELLER (41) wasused for homology modeling of the CRACR2A-a GTPase domain to ahigh-resolution structure of a GPPNHP (5′-guanylyl imidodiphosphate)–bound Rab3a (Protein Data Bank ID: 3RAB). A magnified view of theGPPNHP-binding site is also shown. GPPNHP and the side chains of res-idues important for GTP binding and hydrolysis (Thr559, Gln604, andAsn658) are shown in stick representation. A loop consisting of residues561 to 570 was removed for clarity. Panels were generated with PyMOL(version 1.5.0.4; Schrödinger, LLC). (C) Left: Intrinsic GTP hydrolysis ac-tivity was measured with the indicated FLAG-tagged CRACR2A-a pro-teins that were immunoprecipitated from transfected human embryonickidney (HEK) 293 cells. Data are means ± SEM of triplicate samples froma single experiment and are representative of three experiments. Right:That each sample contained similar amounts of proteins was verified by West-

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ern blotting (WB) analysis of the immunoprecipitates with an anti-FLAG anti-body. WT, wild type; Pi, inorganic phosphate. (D) Left: Jurkat cells transfectedwith plasmids encoding green fluorescent protein (GFP)–tagged WT and theindicatedmutant CRACR2A-a proteins were analyzed by confocalmicroscopyto examine their subcellular localization. White circles mark the cell periphery.Images are representative of three experiments. Scale bar, 5 mm. Right: Densi-tometric analysis (in the indicated numbers of cells) of relative fluorescence in-tensity of GFP in the Golgi compared to that in the cytoplasm. The Golgi wasdetected by staining for endogenous Rab8a. Data are representative of threeexperiments. See fig. S4 for images with Rab8a staining. (E) Jurkat cells stablyexpressing control (Scr) or CRACR2A-specific shRNAwere left unstimulated orwere stimulatedwith anti-CD3 antibodies for indicated times and then analyzedbyWesternblottingwithantibodiesagainst the indicatedproteins.Westernblotsare representativeof three independent experiments.Right:Densitometric anal-ysis of relative band intensities. Data aremeans ± SEMof three independentexperiments. See fig. S5A for the detection of PLC-g1, Lat, ERK, and IkB (in-hibitor of nuclear factor kB). (F) Jurkat cells transfected with control (Scr) orCRACR2A-specific shRNA (R2A) and CRACR2A-depleted Jurkat cells thatwere transfected with plasmids encoding shRNA-resistant WT CRACR2A-a(+a), WT CRACR2A-c (+c), or the indicated mutant CRACR2A-a proteinswere stimulated with anti-CD3 antibody for 10min (left) or kept under restingconditions (right). Cell lysateswere thenanalyzedby intracellular flowcytom-etry with antibodies specific for p-JNK proteins. Data are means ± SD of thepercentages of p-JNK–positive cells from three independent experiments.N.S., not significant. *P < 0.05, **P < 0.005, ***P < 0.0005.




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not substantially affect the phosphorylation of TCR-proximal signalingmolecules, such as PLC-g1 and Lat, or of downstream signaling molecules,including extracellular signal–regulated kinase (ERK) or p38, we observeda marked reduction in the extent of JNK1/2 phosphorylation in these cells(Fig. 1E and fig. S5A). These results suggest that CRACR2A-a plays a rolein selective signaling pathways. To determine whether this function wasshared by other CRACR2A isoforms, we examined the effect of reconstitu-tion of cellswith different isoforms ofCRACR2Aon JNKphosphorylation.Only reconstitution with CRACR2A-a rescued the defect in JNK phospho-rylation in the CRACR2A-deficient cells (Fig. 1F), suggesting thatCRACR2A-a is distinct among CRACR2A proteins in modulating theJNK pathway.

To examine how GTP binding influenced JNK activation byCRACR2A-a, we transfected CRACR2A-deficient Jurkat cells withplasmids encoding CRACR2A-a mutants that were resistant to shortinhibitory RNA. In response to TCR stimulation, cells expressing theGTP-bound Q604L mutant showed enhanced JNK phosphorylationcompared to that in cells expressing the wild-type protein, whereas cellsexpressing the GDP-bound T559N mutant did not show any rescue inJNK phosphorylation (Fig. 1F). These results suggest that GTP binding andGolgi localization are required for the activation of JNK by CRACR2A-a.Under resting conditions, only the constitutively active GTP-boundQ604L mutant CRACR2A protein substantially enhanced JNK phos-phorylation compared to that in cells expressing the wild-type protein(Fig. 1F).

The JNKpathway plays a broad role in T cell activation, including in theproduction of cytokines [for example, interleukin-2 (IL-2)] and the expres-sion of T cell activationmarkers (for example, CD69) through the activationof theAP1 transcription factors (28). To examine the long-termoutcomes ofJNK signaling, we measured the cell surface abundance of CD69 inCRACR2A-depleted Jurkat cells that expressed short hairpin RNA(shRNA)–resistant wild-type or mutant CRACR2A-a proteins. CRACR2A-depleted Jurkat cells had less cell surfaceCD69 than did control cells, whichwas rescued by wild-type CRACR2A-a, but not CRACR2A-c or theT559N mutant CRACR2A-a (fig. S5B), similar to data obtained fromexperiments examining JNKphosphorylation. TheGTP-boundQ604Lmu-tant that mediated enhanced JNK phosphorylation after short-term stimula-tion of the Jurkat cells through the TCR did not rescue the cell surfaceexpression of CD69 after long-term stimulation; instead, it showed adominant-negative effect (fig. S5B). This effect may have been causedby the induction of negative feedback on the AP1 signaling pathway be-cause of excessive JNK activation.

In addition to the mutations that affected GTP binding, we alsochecked the effect on the localization and function of CRACR2A-aof a mutation within the Ca2+-binding EF-hand domain (97DAD99>AAA),which abolishes Ca2+ binding by the CRACR2A-c isoform and enhancesits interaction with the Orai1-STIM1 complex (22). However, the extent oflocalization of the EF-hand mutant of CRACR2A-a to the Golgi-proximalarea was similar to that of the wild-type protein, and it rescued JNK phos-phorylation when expressed in CRACR2A-depleted cells (fig. S5, C andD). Therefore, the Ca2+-sensing function of CRACR2A-a did not seemto play a role in its subcellular localization or its ability to activate JNKunder the conditions tested. Together, these results not only indicate thepresence of CRACR2A-a in addition to CRACR2A-c in a human T cellline but also suggest that CRACR2A-a is involved in regulating both theCa2+ and the JNK signaling pathways, whereas the role of CRACR2A-cappears to be limited to facilitating Ca2+ signaling. Furthermore, these re-sults suggest that the CRACR2A-a–mediated phosphorylation of JNKrequired CRACR2A-a to be localized to the Golgi membranes, whichwas dependent on GTP binding.

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Loss of CRACR2A impairs both the Ca2+-NFAT and JNKsignaling pathways in primary mouse T cellsThe current mouse genomic database annotates only one CRACR2A iso-form: CRACR2A-c (NM_001033464.3); however, Western blotting analy-sis of murine tissues indicated the presence of CRACR2A-a, which wasparticularly abundant in the spleen and lymph nodes (Fig. 2A). To under-stand the structure of the gene encoding mouse CRACR2A-a, we cloned itscomplementary DNA (cDNA) with primers homologous to the humancDNA. Amino acid sequence alignments between the human and mouseCRACR2A-a proteins (deduced from the cloned cDNA sequence) showed80.1% sequence identity and 91.4% sequence similarity (fig. S6A). On thebasis of the cDNA sequence information, we generated an intron-exonmapencoding murine CRACR2A-a (fig. S6B).

We thus designed a targeting vector to delete exons 3 and 4 to abolish theexpression of bothCRACR2A isoforms, considering their redundant role inCa2+-NFAT signaling (Fig. 2B and fig. S6C). CRACR2Afl/fl; CD4Cremicewere generated to ablate CRACR2A expression in the T cell lineage, whichwas validated byWestern blotting analysis (Fig. 2B). Consistent with a rolefor CRACR2A proteins in Ca2+ signaling in Jurkat cells, CRACR2A-deficient naïve CD4+ T cells showed a substantial decrease in the extentofCa2+ entry after TCR stimulation and in passive store depletionwith thap-sigargin (Fig. 2C). Expression of the gene encoding NFATc1, but not thatencoding NFATc2, is induced upon TCR stimulation in a Ca2+-NFATpathway–dependent manner (29). Accordingly, we observed the reducedinduction of NFATc1 protein production in CRACR2A-deficient naïveT cells upon TCR stimulation, whereas the abundance ofNFATc2 remainedunchanged, whichwas suggestive of a role for CRACR2A in long-term reg-ulation of the Ca2+-NFAT pathway (Fig. 2D). Furthermore, we observed asubstantial reduction in the extent of JNK phosphorylation in CRACR2A-deficient naïve CD4+ T cells upon TCR stimulation (Fig. 2E). Impaired ac-tivation of the Ca2+-NFATand JNK signaling pathways led to reduced IL-2production by CRACR2A-deficient cells (fig. S7, A and B), which isconsistentwith results from the experiments involvingCRACR2A-depletedJurkat cells. These observations from the analyses of CRACR2A-deficientprimary CD4+ T cells support our findings from earlier experiments withCRACR2A-depleted Jurkat cells that demonstrated roles for CRACR2Aproteins in the Ca2+-NFAT and JNK signaling pathways.

CRACR2A-a translocates from the Golgi-proximal area tothe immunological synapse through subsynapticvesicles that are distinct from those containing LatNext, we examined how the Golgi-resident CRACR2A-a plays an activerole in TCR signaling. To examine whether CRACR2A-a underwent trans-location from the Golgi to the immunological synapse where signalingmolecules cluster to activate the JNKpathway, we performed confocalmi-croscopic analyses of T cells stimulated on anti-CD3 antibody–coatedcoverslips. Upon TCR stimulation with anti-CD3 antibody, endogenousCRACR2A-a was translocated in vesicles from the Golgi-proximal areato the central area of the cell-coverslip contact site (Fig. 3A and movieS1). Furthermore, incubation of Jurkat cells with Raji cells (a human Bcell line) loaded with superantigen also resulted in the translocationof CRACR2A-a–containing vesicles to the immunological synapse overa short time period, and the entire Golgi was relocated toward the immuno-logical synapse at longer time points (Fig. 3B). In addition, we identified arole for the PRD of CRACR2A-a in the translocation of these vesicles. Weobserved numerous CRACR2A-a–containing vesicles in Jurkat cellsexpressing the DPRD mutant CRACR2A; however, these vesicles failedto accumulate at the immunological synapse (Fig. 3B). These data suggestthat the PRD region may be important for protein-protein interactions thatare essential for the recruitment of CRACR2A-a–containing vesicles to the




immunological synapse. The defect in the translocation of theDPRDmutantalso affected downstream signaling. Compared to CRACR2A-depletedcells expressing wild-type CRACR2A-a, those expressing the DPRD mu-tant did not exhibit substantial rescue of JNK phosphorylation or CD69 ex-pression at the cell surface (Fig. 3C).

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It is generally thought that plasma membrane–resident TCR signalingmolecules cluster at the immunological synapse to activate downstreamsignaling pathways; however, a study found that subsynaptic vesicles alsoplay a role in the recruitment of signaling molecules to the immunologicalsynapse. Themost well-studied vesicles are those bearing Lat, translocation

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Fig. 2. CRACR2A deficiency impairs activa-tion of the Ca2+-NFAT and JNK signalingpathways in primary T cells. (A) Lysates ofthe brain, heart, lung, lymph node (LN), skel-etal muscle (Sk M), and spleen (Spl) ofC57BL/6J mice were analyzed by Westernblotting with antibody (Ab) against CRACR2A-a(R2A-a); b-actin was used as a loading control.Western blots are representative of three ex-periments. (B) Left: Schematic of the targeting

+

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strategy used to delete exons 3 and 4 of mouse CRACR2A. See fig. S6C for the detailed targeting strategy. Right: CD4 T cells from control (WT,CRACR2Afl/fl; Cre−) and knockout (KO, CRACR2Afl/fl; CD4Cre) mice were cultured under nonpolarizing conditions for 4 days and then were analyzedby Western blotting with antibodies against the indicated proteins. (C) WT and CRACR2A-deficient (KO) naïve T cells were subjected to anti-CD3–mediated cross-linking (aCD3 xlink) of the TCR, and intracellular Ca2+ mobilization was then observed. Left: Averaged (± SEM) SOCE responsesfrom control (n = 81) and CRACR2A-deficient (n = 66) CD4+CD25− naïve T cells after stimulation with anti-CD3 antibody and, subsequently, iono-mycin (Iono) in the presence of external solution containing 2 mM Ca2+. Right: Averaged (± SEM) SOCE responses from control (n = 73) andCRACR2A-deficient (n = 76) CD4+CD25− naïve T cells after passive store depletion with thapsigargin in the presence of external solution containing0.5 or 2.0 mM Ca2+ as indicated. Bar graphs show means ± SEM from three independent experiments. (D) NFATc1 and NFATc2 expression in WTand CRACR2A-deficient naïve mouse CD4+ T cells that were left unstimulated or were stimulated with anti-CD3 and anti-CD28 antibodies for theindicated times and then were analyzed by Western blotting with antibodies against NFATc1 and NFATc3. b-actin was used as a loading control. Right:Densitometric analysis of relative band intensities. Data are means ± SEM of three independent experiments. (E) Left: WT and CRACR2A-deficient Tcells were stimulated with anti-CD3 antibody for the indicated times and then were analyzed by flow cytometry to detect p-JNK protein. Right: Theline graph shows average ± SD of the percentage of T cells that were positive for p-JNK from three independent experiments. *P < 0.05, **P < 0.005,***P < 0.0005.

g 22 March 2016 Vol 9 Issue 420 ra31 5



of which is regulated by the vesicle docking protein VAMP7 (14). There-fore, we investigated whether the CRACR2A-a–containing vesicles alsocontained Lat as cargo. However, we could not detect any substantial colo-calization between Lat andCRACR2A-a–containing vesicles in Jurkat cellsstimulated on coverslips coated with anti-CD3 antibody (Fig. 3D). We alsoanalyzed the accumulation of Lat- and CRACR2A-a–containing vesicles atplasma membrane–proximal regions by total internal reflection fluores-

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cence (TIRF) microscopy. TIRF microscopic imaging of cells expressingLat-GFP showed Lat molecules prelocalized at the plasma membrane andtranslocated Lat molecules contained within subsynaptic vesicles (Fig. 3E);however, we did not observe any substantial colocalization between Lat-containing vesicles and CRACR2A-a–containing vesicles (Fig. 3E andmovie S2). These results suggest that the CRACR2A-a–containing vesiclesare distinct from the subsynaptic vesicles that bear Lat.

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Fig. 3. CRACR2A-a translocates from the Golgi to the immunological syn-apse through subsynaptic vesicles that are distinct from those containingLat. (A) Representative confocal images showing localization of endoge-nous CRACR2A-a in a Jurkat cell allowed to spread on stimulatory anti-CD3 antibody–coated coverslip for 10 min. The Golgi was labeled withWGA-594. Individual confocal sections at indicated depths from the bottomof the cell are shown. White boxes (inset) highlight the regions magnified inthe images on the right. Scale bar, 2 mm. Images are representative of17 cells from three experiments. See movie S1 for three-dimensional (3D)projection images. (B) Analysis of the localization of Vav1-GFPwithmCherry-fused WT and DPRD mutant of CRACR2A-a at the immunological synapseformedbetweenJurkat cells and staphylococcal enterotoxin E (SEE)–pulsedRaji cells. Left-most images show theoverlap of bright-field andGFP images.Asterisks in the images show the positions of the Raji cells. The top image isof a Jurkat cell incubated with a Raji cell for 10 min, whereas the middle andbottom images are of Jurkat cells incubated with Raji cells for 20 min. Whiteboxes (inset, 1 to 3) highlight the immunological synapse,which ismagnifiedin the numbered images on the right. Scale bar, 5 mm (unless otherwise in-dicated). Images are representative of 18 cells (for WT) and 12 cells (for theDPRDmutant) from three experiments. (C) Jurkat cells transfected with con-
trol (Scr) or CRACR2A-specific shRNA (R2A) and CRACR2A-depletedJurkat cells that were transfected with plasmids encoding shRNA-resistantWT CRACR2A-a (+WT) or the DPRD mutant (DPRD) CRACR2A-a proteinwere stimulated with anti-CD3 antibody for 10 min and then were examinedby intracellular staining and flow cytometry to detect p-JNK1/2 (left) or werestimulated with anti-CD3 and anti-CD28 antibodies for 18 hours and thenwere examined by flow cytometry to examine the cell surface expressionof CD69 (right). Data are means ± SD from three independent experiments.**P < 0.005. (D) Transfected Jurkat cells expressing Lat-GFP (green) andmCherry (mCh)–CRACR2A-a (red) were allowed to spread on anti-CD3antibody–coated coverslips, and their localization at the contact site and1.4 mmaway from the contact site were determined by confocalmicroscopy.Data in images are averagePearson correlation coefficient values±SEMof13 cells from three experiments. Scale bar, 5 mm (unless otherwise indicated).(E) Jurkat cells expressing Lat-GFP (green, top) and mCherry-fusedCRACR2A-a (red, middle) were imaged by live-cell TIRF microscopy atthe contact site between the cells and anti-CD3–coated coverslips. Bottom:Data in merged images show the average Pearson correlation coefficientvalues ± SEM of 11 cells from four experiments. Scale bar, 5 mm. See movieS2 for time-lapse images of the same cell.




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The recruitment of CRACR2A-a–containing subsynapticvesicles to the immunological synapse depends on aninteraction with Vav1To elucidate the mechanism by which the PRD was involved in the recruit-ment of CRACR2A-a–containing vesicles to the immunological synapse,we investigated possible protein-protein interactions between CRACR2A-aand the Src homology 3 (SH3) domain–containing proximal TCR signalingmolecules that have a high affinity for proline-rich sequences. These effortsled to the identification of Vav1 as an interacting partner of the PRD regionof CRACR2A (Fig. 4A). Among the Ca2+-binding, coiled-coil, proline-rich, and GTPase domains, the PRD showed the highest binding affinityfor Vav1 in pull-down assays with glutathione S-transferase (GST)–fusedproteins. Conversely, we also determined the domainwithin Vav1 that inter-acted with CRACR2A-a. GST pull-down experiments with lysates of cellsexpressing either wild-type CRACR2A-a or its DPRDmutant upon incuba-tionwith aGST-fusedVav1-C fragment (which contains the SH3-SH2-SH3domains in the C terminus of Vav1) showed that Vav1-C interacted withCRACR2A-a, an interaction that was substantially reduced by truncationof the PRD (Fig. 4B). These results suggest that the PRD of CRACR2A-a binds to Vav1. These results were further validated by immuno-precipitation experiments with endogenous proteins, which showed thatthe interaction betweenCRACR2A-a andVav1 in Jurkat cellswas enhancedafter TCR stimulation (Fig. 4C).

Many TCR signaling molecules transiently associate and dissociate atthe immunological synapse (30). To determine the association and dissoci-ation kinetics of CRACR2A-a andVav1, and to elucidate the role ofVav1 inthe recruitment of CRACR2A-a–containing vesicles to the immunologicalsynapse, we performed real-time TIRF microscopy experiments. Wedropped Jurkat cells expressing Vav1-GFP and mCherry-fused wild-typeor DPRD mutant CRACR2A-a on coverslips coated with anti-CD3 anti-body and monitored the recruitment of vesicles to the contact site. Associ-ation and dissociation phenomenawere analyzedwith a double-exponentialwaveformmodel to extract the time constants (Fig. 4D). Vesicles containingCRACR2A-a associatedwithVav1 at the central region of the contact site atvery early times (t1/2 = 33 s; Fig. 4D and movie S3). At later times, Vav1accumulated in the periphery of the cell, whereas the CRACR2A-a–containing vesicles were localized near the central region of the contact site(Fig. 4D). The association of vesicles containing the DPRD mutant withVav1 had similar kinetics to those of vesicles containing the wild-type pro-tein (t1/2 = 37 s), but the extent of the association was diminished (as indi-cated by the reduced value of the Pearson correlation coefficient), and theirdissociation from Vav1 occurred with faster kinetics compared to those ofvesicles containing wild-type CRACR2A (t1/2 = 144 and 312 s for DPRDand wild-type CRACR2A-a, respectively; Fig. 4D and movie S4).

In addition to investigating the DPRDmutant, we also examined wheth-er the accumulation of the Ca2+ binding–defective 97DAD99>AAA mutantCRACR2Aprotein invesicleswas affected. Upon stimulation on coverslipscoated with anti-CD3 antibody, Jurkat cells expressing the 97DAD mutantCRACR2A-a protein accumulated at the contact sites similar to thoseexpressing the wild-type protein (fig. S8A). Together with previous resultsfrom experiments in which JNK phosphorylation was rescued (fig. S5D),these data suggest that a defect in Ca2+ sensing by CRACR2A-a does notalter its translocation in vesicles and its ability to activate JNK under theseconditions.

Next, we examined the effect of Vav1 on the accumulation ofCRACR2A-a–containing vesicles in cells on anti-CD3 antibody–coatedcoverslips. Here, we used ZAP70 as a marker for the contact sites. InVav1-deficient Jurkat cells (known as J.Vav1 cells), the accumulation ofCRACR2A-a–containing vesicles at the contact siteswasmarkedly reducedcompared to that in control cells (Fig. 4E). Furthermore, the loss of Vav1

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substantially decreased the rate of recruitment and the number ofCRACR2A-a–containingvesicles at the antibody-coverslip contact sites (Fig. 4F).Collect-ively, these data from experimentswith theDPRDmutant andVav1-deficientcells suggested an essential role for Vav1 in the recruitment of CRACR2A-a–containing subsynaptic vesicles to the immunological synapse. Vav1 acts as aGEF for Rac1 at the immunological synapse; however, through its C-terminalSH3 and SH2 domains, Vav1 also assembles signaling complexes as a scaf-fold protein independently of its GEF activity (31). It is unlikely that Vav1acts as aGEF forCRACR2A-a, becauseCRACR2A-a predominantly existsin a GTP-bound form at the Golgi. Therefore, the function of Vav1 as asignaling adaptor or scaffold protein may be important for the recruitmentof CRACR2A-a–containing vesicles from the Golgi-proximal area to theimmunological synapse.

In T cells, theCRACchannel componentsOrai1 andSTIM1 cocluster atthe immunological synapse upon contact with APCs (32, 33). BecauseCRACR2A-a also translocated to the immunological synapse and restoredSOCE in CRACR2A-depleted Jurkat cells (fig. S1, B and C), we examinedits possible colocalization with CRAC channel components. TIRF imagingof Jurkat cells expressing STIM1-YFP (yellow fluorescent protein) andmCherry–CRACR2A-a that were dropped on anti-CD3 antibody–coatedcoverslips showed substantial colocalization of both proteins (fig. S8B).Furthermore, passive depletion of ERCa2+ stores by thapsigargin also stim-ulated the translocation of CRACR2A-a–containing vesicles from theGolgi-proximal area to being in close proximity with STIM1 clusters, albeitto a lesser degree than was caused by TCR stimulation (fig. S8C). Theseexperiments suggest that both passive and active store depletion stimulatesthe colocalization of CRACR2A-a–containing vesicles with STIM1 to me-diate CRAC channel activation.

Dissociation of CRACR2A-a from membranes bydeprenylation or GTP hydrolysis stimulatesits degradationSmall RabGTPases associatewith themembranes of theGolgi and vesiclesdepending onwhether GTP is bound and on C-terminal prenylation (17). APrenylation Prediction Suite (PrePS) predicted the attachment of a 20-carbon geranylgeranyl moiety to the C-terminal di-Cysmotif of CRACR2A-a with a high confidence score (E = 8.9 × 10−63; Fig. 5A). However, theC-terminal CCG residues of CRACR2A-a do not constitute a conventionalprenylationmotif (CxCorCC,where x is any amino acid) for RabGTPases.To determine whether this CCG motif was indeed geranylgeranylated andthus important for the retention of CRACR2A-a in the Golgi, we truncatedthese three amino acids togenerate theDC729mutantCRACR2A-a protein.The deletion of these three residues resulted in the predominantly cyto-plasmic localization of the mutant protein, which suggests the possibleprenylation-induced association of CRACR2A-awith the plasma membrane(Fig. 5A). Furthermore, the DC729 mutant CRACR2A-a protein could notrescue JNK phosphorylation in CRACR2A-depleted Jurkat cells, whichsuggests a role for prenylation in this function.

We next examined how treatment with atorvastatin, an inhibitor of pre-nyl transferases, affected the function of CRACR2A-a and compared it witha known target of statins. Treatment of Jurkat cells with atorvastatin sub-stantially decreased the localization ofRac1, a positive control for atorvastatintreatment, to the Golgi membrane (Fig. 5B and fig. S9A). Under the sameconditions, we observed a marked decrease in the extent of the Golgi mem-brane association of CRACR2A-a. We also noticed a marked decrease inthe fluorescence intensity of GFP-tagged CRACR2A-a in atorvastatin-treated cells (Fig. 5B). Consistent with this observation, the abundance ofCRACR2A-a protein was decreased in atorvastatin-treated cells, whereasthat of Rac1 was not substantially changed (Fig. 5C). These results suggestthat the lack of prenylation may induce the degradation of CRACR2A-a.




Because deprenylation by atorvastatin reduced the extent of the associ-ation of CRACR2A-a with the Golgi membrane and induced its degrada-tion, we tested the hypothesis that a switch in the binding of GTP for GDP,which is also important for Golgi membrane association, might stimulatethe degradation of CRACR2A-a.We transfectedHEK293T cellswith plas-

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mids encoding FLAG-tagged wild-type CRACR2A-a, the GTP binding–defective T559N mutant, the guanidine binding–defective N658I mutant,or the GTP hydrolysis–defective Q604L mutant of CRACR2A-a from asingle mRNAwith GFP, which were separated by an internal ribosomal en-try site (IRES). We hypothesized that measurement of the abundance of

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Fig. 4. The Vav1–CRACR2A-a interaction is required for the accumulation ofCRACR2A-a–containing vesicles at the immunological synapse. (A) Top: Pur-ified recombinant GST fusion proteins containing the indicated functional do-mains ofCRACR2A-awere incubatedwith the lysates of anti-CD3–stimulatedJurkat cells and then analyzed by Western blotting for endogenous Vav1.Bottom: Ponceau S staining showed similar amounts of recombinant proteinsin each lane. Data are representative of four experiments. CBD,Ca2+-bindingdomain;CCD, coiled-coil domain. (B) Left: Purified recombinantGST–Vav1-C(containing the C terminus of Vav1) was incubatedwith lysates of Jurkat cellsexpressing GFP-fused WT or DPRD mutant CRACR2A-a and then analyzedbyWesternblotting.Right: PonceauSstainingshowed that similar amountsofrecombinant GST fusion proteins were present in each lane. Data are repre-sentative of three experiments. (C) Left: Lysates of resting or anti-CD3–stimulated Jurkat cells were subjected to immunoprecipitation (IP) withanti-CRACR2A antibody and were analyzed by Western blotting to detectthe indicated proteins. Right: Densitometric analyses of Vav1 band intensitynormalized to that of CRACR2A-a. Data are means ± SEM of threeindependent experiments. IgG, immunoglobulin G. (D) Left: CRACR2A-depleted Jurkat cells coexpressing Vav1-GFP and mCherry-fused WT (top)or DPRD mutant (bottom) CRACR2A-a proteins were dropped onto anti-
CD3–coated coverslips and subjected to live-cell TIRF microscopy to imagethecolocalizationofVav1 (green)with theCRACR2A-aproteins (red). Pearsoncorrelation coefficient values (Rr) are shown in individual images. Scale bar,5 mm. Right: Scatter plot shows mean ± SEM Pearson correlation coefficientvalues at the indicated times. Cell numbers are in parentheses. The datawere fitted with a double-exponential waveformmodel, and the correspond-ing 95% confidence interval of the fit (shaded area) was used to analyze theassociation and dissociation kinetics. See movies S3 and S4 for time-lapseimages of Jurkat cells coexpressing Vav1-GFP and mCherry–CRACR2A-a(movie S3) or mCherry-DPRD (movie S4). (E) WT Jurkat cells (Control, left)and J.Vav1 cells (right) expressingmCh–CRACR2A-a (red) and ZAP70-GFP(green) were dropped onto anti-CD3 antibody–coated coverslips and ana-lyzed by live-cell TIRF microscopy to visualize vesicular translocation. Scalebar, 5 mm. Images are representative of three experiments. (F) Quantificationof the translocation of CRACR2A-a–containing vesicles in the Jurkat (Con-trol) and J.Vav1 cells shown in (E). Left: Line graph shows the mean normal-ized fluorescence intensity ± SEM of mCherry–CRACR2A-a in Jurkat controlcells and J.Vav1 cells. Right: Mean numbers of mCherry–CRACR2A-a–containing vesicles per cell at the contact site at the indicated times. Cellnumbers are in parentheses. *P < 0.05, **P < 0.005, ***P < 0.0005.




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Fig. 5. Degradation of CRACR2A-a by atorvastatin, GTP hydrolysis, andTCR stimulation. (A) Top: Sequence alignment of the prenylation sites inthe C termini of CDC42, Rac1, and CRACR2A-a. CRACR2A-a contains di-Cys residues that are potential targets of the type II GGT and positivelycharged residues (++) that can bind to phospholipids. a5 corresponds tothe fifth a-helix in the GTPase domain, and boxes represent positivelycharged amino acids (++) and prenylation sequences. Middle: Representa-tive confocal images of Jurkat cells expressing GFP-fused WT and mutantCRACR2A-aproteinscostained for endogenousRab8a.Whitecirclesmark thecell periphery. Scale bar, 5 mm.Bottom: (Left)Mean relative fluorescence inten-sity±SDofGFP in theGolgi compared to that in thecytoplasm in the indicatednumbers of cells. (Right) Mean ± SD of the percentages of p-JNK–positivecells among Jurkat cells transfectedwith control (Scr) or CRACR2A-specificshRNA (R2A) and CRACR2A-depleted Jurkat cells that were transfectedwith plasmids encoding shRNA-resistant WT CRACR2A-a (+a) or the indi-cated mutant CRACR2A-a proteins after stimulation with anti-CD3 antibodyfor 10 min. Images are representative of three experiments. (B) Top: Con-focal microscopy images of Jurkat cells expressing GFP-tagged Rac1and CRACR2A-a that were treated with the indicated concentrations ofatorvastatin (Ator) for 16 hours. The atorvastatin target Rac1 was used as apositive control. Scale bar, 5 mm. Bottom: Relative fluorescence intensities of

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GFP-Rac1 (left) and GFP–CRACR2A-a (right) in the Golgi (identified byWGA-594staining; see fig. S9 for imageswithWGA-594costaining) comparedto those in the surrounding areas. Data are means ± SD of three independentexperiments. (C) Top:Jurkatcellswere treatedwith the indicatedconcentrationsof atorvastatin for 16 hours and then analyzed by Western blotting with antibo-diesagainst the indicatedproteins.Bottom:Densitometricanalysisof the relativeband intensities in treated cells normalized to those in untreated cells. Data aremeans ± SEMof three independent experiments. (D) Left: HEK 293T cells ex-pressing N-terminally FLAG-tagged WT or the indicated mutant CRACR2A-acDNAs and GFP from an IRES site were lysed and analyzed by Westernblotting to detect the indicated proteins. The Q604L lane was cut out fromthesameblot (splicepoint indicatedbyvertical black line).Right:Densitometricanalysis of the relative amounts of the indicated mutant proteins normalized tothat of WT CRACR2A-a. Data are means ± SEM of three independentexperiments. The y axis contains a break for clear presentation of data. (E) Top:Jurkat cells (left) and naïve mouse CD4+ T cells (right) were stimulated withanti-CD3antibody for the indicated times, andcellular lysateswere then ana-lyzedbyWesternblottingwithantibodiesagainst the indicatedproteins.Bottom:Densitometric analysis of the relative amounts of CRACR2A-a normalized tob-actin in Jurkat cells (left) and naïve mouse CD4+ T cells (right). Data aremeans ± SEM of three independent experiments. **P < 0.005, ***P < 0.0005.




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wild-type or mutant CRACR2A-a protein, when normalized to that of GFP,would give an estimate of the stabilities of the CRACR2A protein becauseboth they and the GFP proteins would be translated from the same mRNA.The GTP binding–defective T559N and N658I mutants were reduced inabundance, whereas the constitutively GTP-bound Q604L mutant proteinwas increased in abundance compared to that of wild-type CRACR2A-a(Fig. 5D). These results suggest that both prenylation and GTP bindingare required for the association of CRACR2A-a with the Golgi membraneand for its stability. To examine the potential physiological effects of thisfinding, we analyzed the abundance of CRACR2A-a protein in Jurkat cellsandmouseCD4+T cells after TCR stimulation,whichwould be expected toinduce GTP hydrolysis. CRACR2A-a was decreased in abundance in bothJurkat cells and mouse T cells after TCR stimulation (Fig. 5E). Together,these results suggest that TCR stimulation or atorvastatin can cause the cy-toplasmic localization of CRACR2A-a and induce its degradation. Further-more, these data also suggest that CRACR2A-a is a target of atorvastatinand that its degradation may contribute to the anti-inflammatory effects ob-served by atorvastatin administration (19–21).

DISCUSSION

Human CRACR2A encodes two validated transcriptional isoforms:CRACR2A-a and CRACR2A-c. The short isoform CRACR2A-c facil-itates CRAC channel function by enhancing the interaction between Orai1and STIM1 (22). The current mouse genome database shows the presenceof only CRACR2A-c. Our analyses validated the presence of the long iso-form CRACR2A-a in murine tissues, particularly in mouse lymphoidorgans. Both of these isoforms share a conserved function in theCa2+-NFATsignaling pathway, but onlyCRACR2A-a plays a role in the activation of theJNK pathway, which emphasizes the importance of the PRD and GTPasedomains in this distinctive function. Furthermore, our studies reveal a pre-viously uncharacterized mechanism for a Rab GTPase in TCR signaling,which includes recruitment in subsynaptic vesicles to the immunologicalsynapse and inactivation by protein degradation. The activation and in-activation of CRACR2A-a consists of resting, effector, and terminationsteps that are controlled by GTP binding and protein degradation (fig.S9B). In resting T cells, CRACR2A-a needed to be bound to GTP andprenylated to be localized at the Golgi. Unlike most small G proteins,CRACR2A-a predominantly exists in a GTP-bound form localized totheGolgi in resting T cells. After TCR stimulation, CRACR2A-a–containingvesicleswere recruited to the immunological synapse through an interactionwith Vav1, which led to the activation of the downstream JNK pathway.

Passive store depletion also induced the accumulation of CRACR2A-a–containing vesicles in close proximity to the ER–plasma membrane junc-tions to activate SOCE. The mechanism by which translocation of thesevesicles is activated by TCR stimulation or store depletion needs furtherinvestigation. Upon TCR stimulation, CRACR2A-a appeared to undergosome sort of posttranslational modification, as evidenced by the detectionof a higher molecular mass band in the lysates of stimulated T cells withthe anti-CRACR2A antibody (Figs. 4C and 5E). The identity and physi-ological relevance of this posttranslational modification of CRACR2A-aremain unclear. Inactivation of CRACR2A-a occurred through GTP hydrol-ysis, which induced its cytoplasmic localization and degradation (terminationstep). Currently, the composition and fate of the CRACR2A-a–containingvesicles remain unknown because of the lack of additional marker mole-cules. Small GTPases bind to guanine nucleotide dissociation inhibitors(GDIs) after GTP hydrolysis occurs, which enables their stabilization in thecytoplasmand eventually their reinsertion into themembrane (16,34). In con-trast to small GTPases, CRACR2A-a was not recycled; rather, it was con-sumed by entering a unidirectional pathway of protein degradation. Our

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studies also demonstrated that atorvastatin disrupted CRACR2A-a stabilityby inducing this unstable, cytoplasmic state through deprenylation. Thehigh sensitivity of CRACR2A-a to atorvastatin may be because of a lackof CRACR2A-a–specific GDI molecules.

Small G proteins, including RhoA, Rac1, and CDC42, are importantregulators of signaling pathways that control vital functions in T cells, in-cluding thymocyte development, cytoskeletal dynamics, gene transcription,and cell cycle progression (35). Small GTPases are present as GDP-boundinactive precursors and are activated by a switch from GDP to GTP to me-diate their downstream effects. CRACR2A-a is distinctive because it existsin a GTP- and Golgi membrane–bound form in resting T cells and is de-graded after GTP hydrolysis. CRACR2A-a resembles the atypical RhoGTPases, including RhoH, which resides in a constitutively GTP-boundstate (36). Hence, it appears that, similar to RhoH, the activity ofCRACR2A-a may be regulated by localization.

The detailed mechanism by which CRACR2A-a activates thedownstream JNKpathway is unclear. Rac1 andCDC42play important rolesin the stimulation of both p38 and JNK. Activation of the JNK pathwayby Rac1 and CDC42 involves their direct interaction with p21-activatedkinase (PAK1) or other MAPK kinase kinases (MAPKKKs) to phospho-rylate MKK4 and MKK7 (10). It would be interesting to know whetherCRACR2A-a acts as a signaling adaptor to directly activate the downstreamJNKpathway. Alternatively, it is also possible that CRACR2A-a is involvedin the trafficking of vesicles that bear other signaling molecules involved inthe activation of JNK as cargo, supporting the conventional role of smallRab GTPases. The v-SNARE protein VAMP7 plays an important role inthe recruitment of Lat-containing subsynaptic vesicles to TCR clusters,and this recruitment appears to be important for the phosphorylation ofLat and the formation of the Lat signalosome (14). Further studies are re-quired to determinewhether the same cellular machinery is used for the trans-location of CRACR2A-a–containing vesicles. The identity and function ofCRACR2A-a–containing vesicles is likely to be an exciting area for futureinvestigation.

More than 60 members of the Rab GTPase family have been identifiedin humans; however, our knowledge of Rab proteins is limited to the scopeof the role of small Rab GTPases in membrane trafficking. Here, we eluci-dated a previously uncharacterized mechanism of a large Rab GTPase inintracellular signaling that is potentially mediated by vesicular traffickingin T cells. Unlike small G proteins, CRACR2A-a contains additional do-mains, including N-terminal EF hands, a coiled-coil domain, and a PRD.We observed an interaction between CRACR2A-a and Vav1 through thePRD of CRACR2A-a, which was important for the recruitment of CRACR2A-a–containing vesicles to the immunological synapse. These data suggestthat domains of CRACR2A-a other than the GTPase domain are importantfor its distinct functions. These results establish a foundation to understandthe function of other large Rab GTPases, such as Rab44 and Rab45, whichhave domain architectures similar to that of CRACR2A-a (fig. S10). Nu-merous gene linkage analyses of acute and chronic myeloid leukemia andmelanoma identified Rab45 [also known as RASEF (RAS and EF-handdomain containing) or FLJ31614] as a potential tumor suppressor (37, 38);however, the molecular mechanism underlying its link to human diseases isnot clearly understood. Therefore, our study of CRACR2A-a may help inuncovering possible roles of these large Rab GTPases in intracellularsignaling and vesicle trafficking.

In summary, our study has identified CRACR2A proteins as an intra-cellular signaling module that bridges two TCR-proximal signaling path-ways, Ca2+-NFAT and JNK, to facilitate T cell activation. Whereas theshort isoformCRACR2A-cwas only involved in the regulation of Ca2+-NFATsignaling, the long isoform (and large Rab GTPase) CRACR2A-awas impor-tant for both the Ca2+-NFAT and the JNK signaling pathways. We found that

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CRACR2A-awas present in subsynaptic vesicles that translocated from theGolgi region to the immunological synapse after ligation of TCRs, whichwas required for the activation of downstream signaling. Furthermore, thefunction of CRACR2A-awas regulated bymembrane dissociation–induceddegradation upon TCR stimulation. Therefore, this study provides aconceptual framework to advance our understanding of the potential rolesof large Rab GTPases in intracellular signaling and their pathologicalmechanisms related to human diseases.

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MATERIALS AND METHODS

ChemicalsFura-2 AM and wheat germ agglutinin (WGA-594) were purchased fromInvitrogen. Thapsigargin, phorbol 12-myristate 13-acetate, and ionomycinwere purchased from EMD Millipore. Brefeldin A was purchased fromeBioscience. Atorvastatin was purchased from Sigma.

Plasmids and cellsFull-length cDNAencoding humanCRACR2A-a (National Center for Bio-technology Information Reference Sequence: NM_001144958.1) wascloned from a Jurkat cell cDNA library (see table S1 for primers) into thepMSCV-CITE-eGFP-PGK-Puro vector with an N-terminal FLAG tag. Thereverse primer was designed in the putative 3′ untranslated region tosequence the endogenous STOP codon. Our clone contains an additionalserine residue at amino acid position 425 within the PRD (codon 5′-AGT-3′). Various mutants of CRACR2A-a were generated by polymerasechain reaction (PCR) amplification and site-directed mutagenesis (see tableS1 for primers). cDNA encoding CRACR2A-a was N-terminally taggedwith a FLAG tag and subcloned into the lentiviral vector FGllF [a gift fromD. S. An, University of California, Los Angeles (UCLA)]. The cDNAs en-codingwild-typeCRACR2A-1 and itsmutantswere also subcloned into theplasmids pC1-mCherry and pEGFP-C1 (Clontech) to generate proteinsfused N-terminally with mCherry or GFP. HEK 293 and Jurkat E6-1 celllines were obtained from the American Type Culture Collection. Raji cells(a human B cell lymphoma cell line) were a gift from S.Morrison (UCLA).Vav1-GFP, ZAP70-GFP, and GFP-Rac1 clones were purchased fromAddgene. The LAT1-CFP (cyan fluorescent protein) clone and J.Vav1 cellswere gifts from L. Samelson (National Institutes of Health). LAT1 cDNAwas excised from the LAT1-CFP clone with Eco RI and Bam H1 and sub-cloned into the plasmid pEGFP-N1.

Western blotting analysisTo analyze tissues byWestern blotting, wild-typemicewere euthanized andperfusedwith phosphate-buffered saline (PBS) before their organswere har-vested. Tissues were snap-frozen, lysed in radioimmunoprecipitation assay(RIPA)buffer [10mMtris-Cl (pH7.5), 1%TritonX-100, 0.1%SDS, 140mMNaCl, 1 mM EDTA, 0.1% sodium deoxycholate, and protease inhibitorcocktail (Roche)], and centrifuged to remove debris. Samples were resolvedby 8 to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Pro-teins were transferred to nitrocellulose membranes and subsequently ana-lyzed by Western blotting with relevant antibodies. To detect NFAT, 5 ×106 Jurkat cells were stimulated with plate-bound anti-CD3 (10 mg/ml;OKT3, National Cancer Institute preclinical repository) and soluble anti-CD28 antibody (10 mg/ml) for the times indicated in the figure legends,harvested in PBS, lysed in RIPA buffer, and centrifuged to remove debris.Primarymouse T cells (5 × 106)were stimulatedwith plate-bound anti-CD3(10 mg/ml) and soluble anti-CD28 antibody (10 mg/ml) for the times indi-cated in the figure legends. Lysates were resolved by 10% SDS-PAGE andanalyzed by Western blotting to detect NFATc1, NFATc2, and b-actin. To

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analyze TCR signaling, 5 × 106 Jurkat cells or primary T cells were stimu-lated with soluble anti-CD3 antibody (10 mg/ml) and cross-linked with anti-mouse or anti-hamster secondary antibody for the times indicated in thefigure legends, and cell pellets were lysed in SDS loading dye. Lysateswereresolved by 10% SDS-PAGE and analyzed by Western blotting to detectproteins of interest. For atorvastatin treatment, 5 × 106 Jurkat cells weretreated with different concentrations of atorvastatin (in dimethyl sulfoxide).Cell pelletswere lysed inRIPAbuffer and centrifuged to removedebris. Lysateswere resolved by 12% SDS-PAGE and analyzed byWestern blotting to de-tect Rac1, CDC42, CRACR2A, and b-actin. The antibodies used were asfollows: anti–b-actin (Santa Cruz Biotechnology, clone I-19), anti-FLAG(Sigma), anti-NFATc1 (BD Pharmingen, #556602), anti-NFATc2 (SantaCruz Biotechnology, #7296), anti–p-ERK (Cell Signaling Technology,#4377), anti-ERK (Cell Signaling Technology, #9102), anti–p-p38 (CellSignaling Technology, #4511), anti-p38 (Cell Signaling Technology,#9212), anti–p-JNK (Cell Signaling Technology, #9255), anti-JNK (CellSignaling Technology, #9252), anti–p-IkB (Cell Signaling Technology,#9246), anti–PLC-g1 (Cell Signaling Technology, #2822), anti–p-PLC-g1 (Cell Signaling Technology, #2821), anti-Rac1 (Cell Signaling Technol-ogy, #2465), anti–p-Lat (Cell Signaling Technology, #3581), anti-Lat (CellSignaling Technology, #9166), and anti-Vav1 (Cell Signaling Technol-ogy, #2502). Polyclonal rabbit antibody to detect CRACR2Awas gener-ated with purified human CRACR2A-c protein (Open Biosystems) andused at a concentration of 0.1 mg/ml. Chemiluminescence images wereacquired with an Image Reader LAS-3000 liquid-crystal display camera(FujiFilm).

shRNA-mediated knockdownpLKO.1 plasmids encoding shRNAs for the depletion of CRACR2Awerepurchased from Open Biosystems (Thermo Fisher). The sequences of theshRNAs are provided in table S1. To generate lentiviruses for transduction,HEK 293T cells were transfected with plasmid(s) encoding the appropriateshRNA and packaging vectors (pMD2.G and psPAX2, purchased fromAddgene) with the calcium phosphate transfection method. Culture mediumwas harvested at 48 and 72 hours after transfection and used to infect Jurkatcells together with Polybrene (8 mg/ml) through the spinfectionmethod. Cellswere selected with puromycin (1 mg/ml) 48 hours after infection.

Single-cell Ca2+ imaging, TIRF microscopy,and confocal microscopyJurkat cells or primary T cells (1 × 106 cells/ml) were loaded with 1 mMFura-2 AM for 30 min at 25°C and then attached to poly-L-lysine–coatedcoverslips. Measurement of the intracellular Ca2+ concentration ([Ca2+]i)was performed essentially as previously described (39). For TIRF analysis,coverslip bottom dishes were coated with anti-CD3 antibody (10 mg/ml,OKT3) at 37°C for 1 hour, washed with PBS, and used for experiments.Cells were resuspended in Ringer’s solution containing 155 mM NaCl,4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, and 5 mMNa-Hepes (pH 7.4), dropped onto anti-CD3–coated coverslips, and eitherused for time course imaging or fixed after stimulation for 10 min with2.5% paraformaldehyde (PFA) at room temperature and used for confocalmicroscopy analysis. TIRF microscopy was performed with an OlympusIX2 illumination system mounted on an Olympus IX51 inverted micro-scope as described previously (22). Acquisition and image analysis, includ-ing measurement of Pearson correlation coefficient, were performed withSlideBook software (Intelligent Imaging Innovations Inc.), and graphswereplotted with OriginPro8.5 (OriginLab). To quantify TIRF intensity acrossdifferent cells, individual regions of interest were selected, and data wereanalyzed as the ratio of fluorescence intensity at each time point (F) to thatat the start of the experiment (F0). Vesicle number quantification was




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performed by automated counting options in ImageJ. For confocal analysis,eGFP (enhanced GFP) and mCherry were excited sequentially on a ZeissLSM invertedmicroscope (Axiovert 100LSM,Carl Zeiss) fittedwith a 63×water immersion objective lens (C-Apochromat 63×/1.2 W Corr, CarlZeiss). Images were acquired and processed for enhancement of brightnessand contrast with Pascal5 software (Carl Zeiss).

GTPase activity assayHEK293T cells were transfectedwith empty plasmid or plasmids encodingwild-type or mutant CRACR2A-a proteins. Forty-eight hours after transfec-tion, the cellswere harvested, lysed in lysis buffer [20mM tris-Cl (pH 7.5),2 mM EDTA, 100 mM NaCl, 10% glycerol, 1.0% Igepal CA-630, andprotease inhibitor cocktail], and centrifuged to remove the debris. Cell ly-sates were then precleared with protein A–Sepharose and subjected to im-munoprecipitation overnight with anti-FLAG antibody–conjugated agarose(Sigma). Immunoprecipitated samples were washed in lysis buffer threetimes and then washed twice more in lysis buffer without EDTA andIgepal CA-630. Immunoprecipitates were directly used for the analysisof GTPase activity with a colorimetric GTPase assay kit (Novus Biologi-cals), and absorbance was read on a POLARstar Omega plate reader(BMG Labtech).

Measurement of JNK phosphorylation byintracellular stainingExponentially growing Jurkat cells (5 × 106) were transfected with emptyvector or plasmids encoding CRACR2A-c and wild-type or mutantCRACR2A-a proteins by electroporation at 200Vwith an ECM830 electro-porator (Harvard Apparatus). Forty-eight hours after transfection, the cellswere left untreated or were stimulated with anti-CD3 antibody (10 mg/ml,OKT3) for 10 min. Naïve CD4+ T cells isolated from control andCRACR2Afl/fl-CD4Cre animals were stimulated with anti-CD3 antibody(1 mg/ml, 1452C11) for the times indicated in the figure legends. Cells werefixedwith 4%PFA, permeabilizedwith ice-coldmethanol, and stainedwithan anti–p-SAPK/JNK monoclonal antibody conjugated with Alexa Fluor647 (Cell Signaling Technology, #9257). Cells were washed twice withPBS and analyzed with a FACSCalibur flow cytometer (Becton Dickinson)and FlowJo software.

Immunological synapse andimmunofluorescence analysisFor immunological synapse analysis, Jurkat cells were electroporatedwith the appropriate plasmids and used for experiments 24 hours later.To visualize Jurkat cells interacting with superantigen-pulsed Raji cells,CMAC (7-amino-4-chloromethylcoumarin) blue dye (Invitrogen)–loadedRaji cells were pulsed with staphylococcal enterotoxin E toxin (1 mg/ml;Toxin Technology) for 45 min in complete medium. After rinsing, the Rajicells were mixed with Jurkat cells at a ratio of 1:1 for 10 or 20 min onpoly-D-lysine–coated coverslips at 37°C, fixed with 2.5% PFA, and rinsedwith PBS. Immune conjugates were examined on an inverted Zeiss LSM(Axiovert 100 LSM) confocal microscope as described earlier. For immuno-fluorescence staining of endogenous CRACR2A, Jurkat cells fixed with2.5% PFA were washed three times with PBS, permeabilized with 0.5%Igepal CA-630, blocked with 2.5% fetal bovine serum, and stained withpurified anti-CRACR2A antibody for 1 hour at room temperature. Thecells were washed with PBS, incubated with fluorescein isothiocyanate–labeled anti-rabbit antibody for 30 min, mounted, and examined on a ZeissLSM confocal microscope. Anti-Rab8a antibody (clone 3G1) was purchasedfrom Abnova, anti-GM130 antibody (D6B1) was purchased from CellSignaling Technology, and anti-Golgin antibody (#A21270) was purchasedfrom Invitrogen.

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GST pull-down analysis and immunoprecipitationsJurkat cells (2 × 107) were harvested in PBS, lysed in lysis buffer, andcentrifuged to remove debris before being precleared with glutathione–Sepharose 4Bbeads. Lysateswere incubatedwith 20mgofGSTorGST-taggedfragments of the Ca2+-binding domain (amino acids 1 to 197), coiled-coildomain (amino acids 198 to 348), PRD (amino acids 349 to 540), or GTPasedomain (amino acids 541 to 731) of CRACR2A-a for 18 hours in bindingbuffer [20 mM tris-HCl (pH 7.5), 100 mMNaCl, 2 mMEDTA, 1.0% IgepalCA-630, protease inhibitors, and 10% glycerol]. Pull-down samples werewashed five times with lysis buffer and analyzed by Western blotting. ForGSTpull-downwith theVav1C terminus,HEK293Tcells (5×106) expressingGFP-taggedwild-typeCRACR2A-a or theDPRDmutant ofCRACR2Awerelysed as described earlier and incubatedwith 20 mg of GSTor the GST-taggedC-terminal fragment of Vav1 (amino acids 637 to 870) overnight as describedearlier. For immunoprecipitations, Jurkat cells (2 × 107) were lysed in lysisbuffer, centrifuged to remove debris, and preclearedwith proteinA–Sepharose.The precleared lysateswere incubatedwith 1 mg of rabbit IgG anti-CRACR2Aantibody for 18 hours. Immunoprecipitates were washed five times in lysisbuffer and analyzed by Western blotting to detect Vav1 and CRACR2A.

Generation of CRACR2A knockout miceTargeting of CRACR2Awas performed by flanking exons 3 and 4 with LoxPsites by homologous recombination in AB2.2 (129SvEv) embryonic stem (ES)cells. Aberrant splicing of exons 2 to 5 causes a shift in the reading frame,whichresults in truncation of the protein. Deletion of exons 3 and 4 results in loss ofexpressionofboth isoformsofCRACR2A.G418-resistant cloneswere screenedby PCR for homologous recombination at both homology arms. Chimericmicewith floxed CRACR2A alleles were generated by blastocyst injection of hetero-zygous CRACR2Afl/+ ES cell clones. Founder CRACR2Afl/+ chimeric micewere bred with Flp-deleter mice (Jackson Laboratory) to remove the neomycinresistancegenecassette.CRACR2Afl/flmicewerebackcrossed toC57BL/6micefor at least 10generations and thenbredwithCD4Cremice (JacksonLaboratory)to generate T cell–specific deletion of CRACR2A. All mice were maintained inpathogen-freebarrier facilities andused in accordancewithprotocols approvedbythe Institutional Animal Care and Use Committee at the UCLA.

T cell purificationT cell purification, activation, and transduction were performed as previouslydescribed (40). CD4+T cellswere purified bymagnetic sorting from single-cellsuspensions generated by mechanical disruption of the spleens and lymphnodes of adult mice according to the manufacturer’s instructions (Invitrogen).For effector T cell differentiation, cells were stimulatedwith anti-CD3 antibody(1 mg/ml; 1452C11, Bio X Cell) and anti-CD28 antibody (1 mg/ml; Pharmin-gen) for 48 hours on plates coated with goat anti-hamster secondary antibodyfor cross-linking the anti-CD3 antibody (0.3 mg/ml; MP Biomedicals).

Statistical analysisStatistical comparisons were performed with a two-tailed Student’s t test.Normal distribution of data was evaluated with the Shapiro-Wilk test, andequal variance was tested with the F test. For all of the tests, P <0.05 wasconsidered to be statistically significant.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/9/420/ra31/DC1Fig. S1. CRACR2A-a plays a role in SOCE in Jurkat cells.Fig. S2. The C terminus of CRACR2A-a contains a Rab GTPase domain.Fig. S3. Subcellular localization of CRACR2A-a in comparison with Golgi markers andsmall GTPases.Fig. S4. Subcellular localization of wild-type and mutant CRACR2A-a proteins.Fig. S5. CRACR2A-a is required for the TCR-stimulated phosphorylation of JNK and cellsurface expression of CD69.




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Fig. S6. Genomic organization of human and murine CRACR2A.Fig. S7. CRACR2A deficiency or depletion reduces cytokine production by T cells.Fig. S8. CRACR2A-a colocalizes with STIM1 after store depletion.Fig. S9. Degradation of CRACR2A-a by atorvastatin.Fig. S10. Members of the large GTPase family are defined by their similar domain architectures.Table S1. List of primers and shRNAs used in this study.Movie S1. Rotation of the 3D projection of endogenous CRACR2A in a Jurkat cell on ananti-CD3 antibody–coated coverslip.Movie S2. TIRF imaging of Lat and CRACR2A-a at the plasmamembrane of a spreading Jurkat cell.Movie S3. TIRF imaging of Vav1 and CRACR2A-a at the plasma membrane of aspreading Jurkat cell.Movie S4. TIRF imaging of Vav1 and DPRD CRACR2A-a at the plasma membrane of aspreading Jurkat cell.

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Acknowledgments: We thank V. Barr and L. Samelson (NIH) for sharing the Lat plasmid, the J.Vav1 cell line, and protocols for the stimulation of Jurkat cells with anti-CD3 antibody and APCs. Wealso thank M. Oh-hora (Kyushu University, Japan) and B. Ribalet for helpful suggestions. Funding:This work was supported by NIH grant AI083432 (Y. Gwack) and American Heart Association grant12SDG12040188 (S.S.). Flow cytometry was performed in the UCLA Jonsson ComprehensiveCancer Center (JCCC) and Center for AIDS Research (CFAR) Flow Cytometry Core Facility, whichissupportedbyJCCCgrantP30CA016042andCFARgrant5P30AI028697.Authorcontributions:S.S. and Y. Gwack designed the research; S.S. cloned and generated CRACR2Afl/fl animals andperformed all the Ca2+ imaging, microscopy, and statin treatment experiments; K.-D.K. performedthe in vitro T cell analysis and JNKexperiments with help from J.S.W.; Y. Gao performed biochemicalexperiments to analyze CRACR2A and Vav1 interactions and performedGTPase assays; S.G. per-formedWestern blotting analysis of CRACR2A andNFAT; G.C. helpedwith statistical analyses; A.P.and J.A. built the structural homologymodel of theGTPase domain of CRACR2A-a;M.J. helpedwiththe generation of theCRACR2Afl/fl mice; andS.S. andY.Gwackwrote themanuscript with input fromthe other authors.Competing interests: The authors declare that they have no competing interests.

Submitted 30 June 2015Accepted 7 March 2016Final Publication 22 March 201610.1126/scisignal.aac9171Citation: S. Srikanth, K.-D. Kim, Y. Gao, J. S. Woo, S. Ghosh, G. Calmettes, A. Paz,J. Abramson, M. Jiang, Y. Gwack, A large Rab GTPase encoded by CRACR2A is acomponent of subsynaptic vesicles that transmit T cell activation signals. Sci. Signal. 9,ra31 (2016).



transmit T cell activation signalsis a component of subsynaptic vesicles that CRACR2AA large Rab GTPase encoded by

Abramson, Meisheng Jiang and Yousang GwackSonal Srikanth, Kyun-Do Kim, Yuanyuan Gao, Jin Seok Woo, Shubhamoy Ghosh, Guillaume Calmettes, Aviv Paz, Jeff

DOI: 10.1126/scisignal.aac9171 (420), ra31.9Sci. Signal.

kinase signaling.CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium andcell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited

T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T+CD4triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human

found that the lymphocyte-specific large guanosineet al.at the site of the T cell receptors (TCRs). Srikanth T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex

Recruiting vesicles to activate T cells

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