Ubc9 Interacts with and SUMOylates the TCR Adaptor SLP-76 for NFAT Transcription in T Cells

Yiwei Xiong, Yulan Yi, Yan Wang, Naiqi Yang, Christopher E. Rudd and Hebin Liu
J Immunol December 1, 2019, 203 (11) 3023-3036; DOI: https://doi.org/10.4049/jimmunol.1900556

Key Points

  • Ubc9 interacts with and SUMOylates the immune adaptor SLP-76 in T cells.

  • Synergy of SLP-76–Ubc9 on IL-2 transcription is SLP-76 SUMOylation dependent.

  • SLP-76 SUMOylation is required for Ubc9-NFAT complex assembly for IL-2 transcription.

Abstract

Although the immune adaptor SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) integrates and propagates the TCR signaling, the regulation of SLP-76 during the TCR signaling is incompletely studied. In this article, we report that SLP-76 interacts with the small ubiquitin-like modifier (SUMO) E2 conjugase Ubc9 and is a substrate for Ubc9-mediated SUMOylation in human and mouse T cells. TCR stimulation promotes SLP-76–Ubc9 binding, accompanied by an increase in SLP-76 SUMOylation. Ubc9 binds to the extreme C terminus of SLP-76 spanning residues 516–533 and SUMOylates SLP-76 at two conserved residues K266 and K284. In addition, SLP-76 and Ubc9 synergizes to augment the TCR-mediated IL-2 transcription by NFAT in a manner dependent of SUMOylation of SLP-76. Moreover, although not affecting the TCR proximal signaling events, the Ubc9-mediated SUMOylation of SLP-76 is required for TCR-induced assembly of Ubc9-NFAT complex for IL-2 transcription. Together, these results suggest that Ubc9 modulates the function of SLP-76 in T cell activation both by direct interaction and by SUMOylation of SLP-76 and that the Ubc9–SLP-76 module acts as a novel regulatory complex in the control of T cell activation.

Introduction

The engagement of T cells with APCs evokes signaling cascades that are crucial to T cell activation and function during an immune response. Recognition of Ag peptide–MHC complex by TCR initiates T cell activation signals, which are orchestrated and fine-tuned by an array of signaling molecules, including specific protein tyrosine kinases and immune adaptors (1–3). Irregularities in the regulation of TCR signaling events are associated with immunodeficiency or autoimmunity diseases (4).

The immune adaptor SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a central regulator of TCR signaling that integrates and propagates signals emanating from the TCR stimulation to their downstream effectors (5–7). SLP-76 contains multiple protein-interacting domains, through which it acts as a scaffold for the recruitment and assembly of multiple signaling molecules required for T cell activation: an N-terminal sterile α motif (SAM) domain that mediates the self-association of SLP-76 (8, 9), a central proline-rich region (PRR) that binds to the SH3 domain of Grb2-related adaptor downstream of Shc (GADS) and phospholipase Cγ1 (PLCγ1) (10, 11), and a C-terminal SH2 domain that interacts with adhesion and degranulation-promoting adapter protein (ADAP) and the hematopoietic progenitor kinase 1 (HPK1) (12, 13). In addition, the three tyrosine motifs (Y113, Y128, and Y145) are phosphorylated by ZAP-70 and mediate the binding to vav guanine nucleotide exchange factor 1 (Vav1), Nck, and IL-2–inducible T cell kinase (Itk) following TCR ligation (14–17). Loss of SLP-76 in mice blocks T cell development at an early stage of double-negative 3 (18). Simple quantitative reductions of SLP-76 are sufficient to cause immune dysregulation (19). SLP-76 also exerts a feedback regulation on the clustering and activity of ZAP-70 (20).

The TCR signaling cascade involves the proximal and distal signaling events. The early proximal events of TCR signaling include activation of protein tyrosine kinases with the resultant phosphorylation of effector molecules, microcluster formation, assembly of proximal signalosome, activation of PLCγ1, calcium influx, etcetera (1–3). The signaling molecules and second messengers further trigger a variety of distal signaling events, such as the activation of NFAT, activation of ERK and NF-κB pathways, cytoskeletal rearrangement, and integrin activation, ultimately leading to new gene transcription, cytokine production, and activation of T cells (1). An increase in the transcription of the cytokine IL-2 is one key consequence of T cell activation controlled by the binding of NFAT and other transcription factors to the IL-2 promoter (21). The classic activation of NFAT is mediated by calcium/calcineurin pathways where an increase in intracellular Ca2+ activates calcineurin, leading to the dephosphorylation and nuclear translocation of NFAT (22, 23). NFAT together with AP-1 binds to the DNA response element IL-2 promoter to induce the gene transcription of IL-2 (24). On another level, we previously showed that SLP-76 binds to SUMOylated Ran GTPase activating protein 1 (RanGAP1) of the nuclear pore complex to facilitate the nuclear import of transcription factor NFAT, pointing to a further downstream contribution of SLP-76 to NFAT-driven gene transcription in the nucleus of T cells (25).

The posttranslational modification with small ubiquitin-like modifier (SUMO), namely SUMOylation, has been implicated in many key cellular processes, including cell division, DNA replication, nuclear transportation, and transcription by fine-tuning protein functionality in many aspects, such as protein–protein interaction, subcellular localization, stability, and activity (26–28). Until recently, regulation of T cell functions by the SUMO pathway had been unexplored. SUMOylation of protein kinase C-θ (PKC-θ) regulates the association of CD28 with PKC-θ and filamin A and is required for the organization of a mature immunological synapse and T cell activation (29). SUMOylation of PLCγ1 facilitates the assembly of PLCγ1 microclusters and promotes its interaction with SLP-76 and GADS (30). Further, loss of Ubc9, the sole SUMO E2 conjugase, leads to defects in T regulatory homeostatic proliferation and suppressive function as well as defective late-stage maturation in the thymus with increased apoptosis and impaired proliferation (31, 32). Our previous work identified Ubc9 as a regulator of TCR-mediated integrin adhesion, by which it functions independently from its enzymatic activity but instead acts as a signaling molecule and interacts with immune adaptor ADAP in the “inside-out” signaling pathway (33). Further, Ubc9 binds to and mediates NFAT SUMOylation, which prolongs the stay of NFAT in nucleus (34, 35). Despite this, a functional link between SUMOylation and TCR signaling has been incompletely studied.

In the current study, we demonstrate that Ubc9 binds to the extreme C terminus of SLP-76, resulting in the SUMOylation of two conserved lysine residues within the PRR of SLP-76. Further, SLP-76 and Ubc9 synergized to potentiate TCR-mediated IL-2 transcription and production in a manner dependent of SLP-76 SUMOylation. Although not affecting TCR proximal signaling events, SUMOylation of SLP-76 is required for the TCR-induced assembly of the Ubc9-NFAT complex for IL-2 transcription. Together, our data indicate SUMOylation as a posttranslational mediator of TCR signaling through Ubc9 interaction and SUMOylation of the key TCR adaptor protein SLP-76.

Materials and Methods

Cell culture and reagents

Jurkat T cells, murine T cell hybridoma DC27.10 cells, and SLP-76–deficient Jurkat J14 T cells (gift from A. Weiss, University of California, San Francisco) were grown in RPMI 1640 medium with 5% (v/v) FBS and 100 U/ml penicillin/streptomycin. HEK293T cells were cultured in DMEM supplemented with 5% (v/v) FBS and 100 U/ml penicillin/streptomycin. Anti-mouse CD3 (2C11) and anti-human CD3 (OKT3) were obtained from BioLegend. Anti-human and anti-mouse CD28 were obtained from eBioscience and BioXCell, respectively. Anti-ADAP and anti-phosphotyrosine (clone 4G10) were purchased from Millipore. Anti-hemagglutinin (HA) and anti-FLAG were from Sigma-Aldrich. Anti–SLP-76, anti-SUMO1, and anti-NFATc1 were from Cell Signaling Technology. Anti-Ubc9, anti–src kinase associated phosphoprotein 1 (SKAP1), anti-GST, and anti-GADS were from Proteintech. Anti-RanGAP1 was from Santa Cruz Biotechnology. Anti–⍺-tubulin and anti-GAPDH were from Abcam.

Constructs, transfection, and luciferase assay

The HA-tagged SLP-76, HA-tagged RanGAP1, HA-tagged ADAP, and HA-tagged SKAP1 have been described previously (25, 33). SLP-76–Ubc9 fusion vector was generated by subcloning the full-length SLP-76 cDNA into the BamHI and EcoRI sites of pcDNA3-MCS-Ubc9 (kind gift from R. Niedenthal, Institut für Physiologische Chemie, Hanover, Germany). Construct of SUMO1 was inserted into a 2× FLAG-tagged pcDNA3.1 vector. Mutants were generated by site-directed mutagenesis using the QuikChange protocol and Canace High-Fidelity DNA Polymerase (Yeasen). The DNA sequences of all plasmid inserts were confirmed by Sanger sequencing.

Transfection of Jurkat T cells was performed with Gene Pulser Xcell Electroporator (Bio-Rad) using 250 V, 800 μF. Stable ADAP-knockdown Jurkat T cells were generated via lentiviral delivery of shRNA (5′-GCAAAGGCCAGACGTCTTA-3′) (36) in a pLKO.1 vector and then selected with puromycin for at least 3 wk before experimental use. For the generation of stable T cells that harbored arginine substitution of the lysine residues K266 and K284, the HA-tagged SLP-76 K266R or K284R constructs were inserted into a pLVX-IRES-Puro Vector and transfected into SLP-76–deficient J14 Jurkat T cells, followed by selection with puromycin for at least 3 wk before use. For transfection of DC27.10 cells, 1 × 107 cells were resuspended in 350 μl of RPMI 1640 medium and mixed with 30 μg of DNA in a 4-mm cuvette. A 260-V, 950-μF pulse was applied to the cuvette using Gene Pulser Xcell Electroporator. After transfection, the cells were cultured in antibiotic-free complete medium for 24 h before analysis. Transfection of HEK 293T cells was performed on 50–80% confluent cells in 10-cm dishes using liposomal transfection reagent (Yeasen) according to the manufacturer’s instructions. For luciferase assay, 5 × 106 of Jurkat T cells were transfected with 5 μg of 3× NFAT-luc or NF-κB-luc reporter plasmid, 5–10 μg of expression vector, and 0.5 μg of pRL-TK Renilla Luciferase Control Plasmid (Promega), followed by anti-CD3 stimulation for 6 h. Luminescence was measured using a microplate reader (PHERAstar FS; BMG LABTECH GmbH). The firefly luciferase activity was normalized to the Renilla luciferase activity, and the fold induction in relative luciferase units of the experimental construct were calculated by dividing the normalized luciferase unit of each experimental construct by those of control vectors under resting condition.

Immunoprecipitation and immunoblotting

Cell extracts were prepared in lysis buffer (1% Triton X-100 [v/v] in 20 mM Tris-HCl [pH 8.3], 150 mM NaCl, 1 mM Na4VO3, and 0.1% protease inhibitor mixture solution [Roche]). For the detection of SUMOylated proteins, 10 mM cysteine peptidase inhibitor iodoacetamide was freshly added to the lysis buffer to preserve SUMO-conjugated proteins. For immunoprecipitation experiments, cell extracts were precleared with 25 μl of Protein G Sepharose Beads (Amersham). Precleared supernatants were then incubated with Abs at 4°C overnight, followed by incubation with 25 μl of Protein G Sepharose Beads at 4°C for another 1 h. Bound beads were washed three times with lysis buffer, and precipitates were eluted from beads by boiling in sample buffer for 10 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA prior to immunoblotting with indicated Abs at 4°C overnight. Secondary Abs were IRDye-labeled anti-mouse and anti-rabbit IgG (LI-COR). Membranes were visualized with Odyssey Imaging Systems (LI-COR).

GST pull-down assay

Sequence encoding residues 516–533 and residues 403–438 of human SLP-76 were amplified by PCR and subcloned into the PGEX-4T-1 to generate GST-SLP-76-516-533 and GST-SLP-76-403-438, respectively. GST fusion proteins were expressed in Escherichia coli BL21 cells and purified using a GST-Tag Protein Purification Kit (Beyotime), following the manufacturer’s instructions. For GST pull-down assay, GST protein, GST-SLP-76-516-533, or GST-SLP-76-403-438 recombinant protein were incubated with recombinant Ubc9 protein (Sino Biological) at 4°C for 1 h and then immobilized on glutathione-agarose beads for 45 min at 4°C. Beads were washed five times with cell lysis buffer and boiled in sample buffer for 10 min. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blotting for the detection of proteins of interest.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed as previously described with minor modifications (37). Briefly, cells (1 × 107) were cross-linked with 1% formaldehyde and quenched by adding glycine to a final concentration of 125 mM. Nuclei were pelleted and resuspended in lysis buffer (50 mM Tris-HCl [pH 8], 10 mM EDTA, 1% SDS) supplemented with protease inhibitor mixture. Lysate was subjected to sonication for six rounds of 15-s pulses followed by 45-s rest periods to yield chromatin fragments ranging between 300 and 1000 bp in size. The sonicated extracts were then incubated with anti-NFATc1 at 4°C overnight. Protein–DNA complexes were recovered using Protein G Sepharose Beads at 4°C for another 2 h. Following four high-salt washes, the complexes were eluted in elution buffer (50 mM Tris-HCl [pH 8], 10 mM EDTA, 1% SDS) supplemented with 20 μg of proteinase K and incubated at 55°C for 2 h. Cross-links were reversed by incubation at 65°C overnight. DNA was purified by phenol/chloroform extraction and used for PCR. The primers specific for the IL-2 promoter were used as previously described (sense: 5′-GTTTCATACAGCAGGCGTTCATTG-3′; antisense: 5′-TTTCCTCTTCTGATGACTCTCTGG-3′) (38).

Quantitative RT-PCR

Total RNA was isolated from the cells using TRI-reagent (Sigma-Aldrich) and reverse transcribed using RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific), following the manufacturer’s manual. Quantitative PCR analysis was performed using the SYBR Green master mix (Yeasen) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Primer sequences used were as follows: IL-2 sense 5′-CCTGAGCAGGATGGAGAATTACA-3′ and antisense 5′-TCCAGAACATGCCGCAGAG-3′ (39). Quantification was normalized to β-actin gene. All PCR reactions were performed in triplicate.

ELISA

Mouse splenocytes were freshly isolated from 6- to 8-wk-old C57BL/6 mice (Shanghai Laboratory Animal Center, Chinese Academy of Sciences). All animal studies were approved by the Animal Ethics Working Group at Xi’an Jiaotong-Liverpool University and comply with the animal management regulations of China for the care and uses of experimental animals. Cells were transfected with indicated plasmids using the Amaxa Nucleofector kit, following the manufacturer’s instructions. Twenty-four hours after transfection, cells were either left unstimulated or activated with 5 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for another 48 h. Cell supernatants were collected, and the levels of IL-2 in the supernatant were measured with a commercially available ELISA kit (Dakewe), according to the manufacturer’s manual.

Nano liquid chromatography–tandem mass spectrometry

Immunoprecipitates were prepared as described in Immunoprecipitation and immunoblotting and resolved on an 8% SDS polyacrylamide gel. Gel was then stained by GelCode Blue staining solution (Thermo Fisher Scientific) and was excised, digested with trypsin, and analyzed by Nano liquid chromatography–tandem mass spectrometry (LC-MS/MS), following a previously described protocol (40). Samples were loaded and eluted into a surveyor liquid chromatography system (Shimadzu LC-20AD) online, coupled to a Linear Trap Quadropole Orbitrap Velos (Thermo Fisher Scientific). Spectra data were searched against the human UNIPROT nonredundant protein database using Mascot v2.3 (Matrix Science). The database search used the following options: trypsin enzyme specificity, one possible missed cleavage, 20 ppm mass tolerance for peptide ions, 0.1 Da mass tolerance for fragment ions, “Carbamidomethyl (C)” for fixed modifications, and “Oxidation (M); Gln->pyro-Glu (N-term Q); Deamidated (NQ); Ubiquitination (K); GlyGly (K)” for variable modifications. Mascot results were filtered at 1% false discovery rate at peptide level.

Confocal imaging

For live cell imaging on microcluster formation, Jurkat T cells transfected with EYFP–SLP-76 wild type (WT), EYFP–SLP-76 mutants, and/or mRFP-Ubc9 constructs were imaged on poly-l-lysine– and anti-CD3–coated chambered coverslides (LabTek) at 37°C, 5% CO2, as previously described (20). The poly-l-lysine–coated coverslides were treated with anti-CD3 (OKT3, 10 μg/ml) for 1 h at 37°C and blocked for 1 h in 1% BSA. Cells were plated on the coverslides and imaged using a Zeiss 880 confocal microscope with a 63× Plan-Apochromat oil immersion objective. Images were acquired with Zen software and processed by Image J. The colocalization analysis of SLP-76 and Ubc9 was performed by calculating the Pearson correlation coefficients using a Pearson-Spearman correlation colocalization plugin of Image J, as previously described (41).

EMSA

Cells transfected with empty vector, SLP-76 WT, or mutants were stimulated by anti-CD3 and anti-CD28 for 1 h. Nuclear fractions were extracted, and protein amount was determined by bicinchoninic acid protein assay (Pierce). Ten micrograms of proteins were incubated with Cy3-labeled IL-2 (−90) NFAT probe (sense: 5′-CTCTTTGAAAATATGTGTAATATGTAAAACAT-3′; antisense: 5′-ATGTTTTACATATTACACATATTTTCAAAGAG-3′), IL-2 (−90) NFAT mutant probe with mutations at NFAT consensus binding site (sense: 5′-CTCTTTTTCCATATGTGTAATATGTAAAACAT-3′; antisense: 5′-ATGTTTTACATATTACACATATGGAAAAAGAG-3′), IL-2 (−45) NFAT probe (sense: 5′-CACCCCCATATTATTTTTCCAGCATT-3′; antisense: 5′-AATGCTGGAAAAATAATATGGGGGTG-3′), or IL-2 (−45) NFAT mutant probe (sense: 5′-CACCCCCATATTATTTTTAAAGCATT-3′; antisense: 5′-AATGCTTTAAAAATAATATGGGGGTG-3′) (42) before being separated by electrophoresis and visualized in ChemiDoc MP Imaging System (Bio-Rad).

Statistical analysis

Statistical analysis was performed in GraphPad Prism 7 software with unpaired Student t test or the Mann–Whitney U test for nonparametric data when comparing differences of means between two groups. For the comparisons of more than two groups, a one-way or two-way ANOVA followed by correction for Bonferroni multiple comparison test was used. The p values are as follows: *p < 0.05 and ****p < 0.0001.

Results

Ubc9 potentiates TCR-induced IL-2 transcription by NFAT

In a previous study, we have showed that Ubc9 regulates integrin-mediated T cell adhesion (33). To find out whether Ubc9 also plays a role in TCR-mediated T cell activation, we first assessed the effect of Ubc9 overexpression on TCR activation by the IL-2 promoter luciferase reporter assay. As shown in Fig. 1A, upon anti-CD3 stimulation, overexpression of Ubc9 increased the NFAT-driven IL-2 promoter activity by ∼1.8-fold when compared with empty vector controls but had no obvious effect on NF-κB–driven promoter activity.

FIGURE 1.
FIGURE 1.

Ubc9 potentiates TCR-induced IL-2 transcription by NFAT. (A) Ubc9 enhances NFAT-driven IL-2 promoter activity. Jurkat T cells were cotransfected with either empty vector or Ubc9 constructs plus luciferase reporter plasmid containing NFAT-targeted sequence or NF-κB–targeted sequence from the IL-2 promoter and pRL-TK plasmid (left). The expression of Ubc9 was assessed by Western blotting using anti-Ubc9 (right). Data are represented as mean ± SEM from five experiments. (B) Ubc9 increases NFAT binding activity to IL-2 promoter. Mouse T cell hybridoma DC27.10 cells were transfected with empty vector or Ubc9 constructs, followed by stimulation with or without anti-CD3 plus anti-CD28 for 3 h. The IL-2 promoter was amplified with PCR from the precipitated DNA by anti-NFATc1 Abs or from sheared chromatin taken prior to immunoprecipitation as input control. One representative of three independent experiments was shown. (C) Immunoblot analysis of total tyrosine phosphorylation, p38, ERK1/2, and AKT phosphorylation in Jurkat T cells expressing either empty vector or Ubc9 constructs followed by stimulation with or without anti-CD3 for 3 min (left). Histogram showing the ratio of densitometry value of protein bands and normalized to the results obtained in resting cells expressing empty vectors in the same experiment (right). Data are represented as mean ± SEM from at least three experiments. (D) Ubc9 potentiation in IL-2 transcription is impaired in SLP-76–deficient J14 cells. Jurkat T cells and J14 Jurkat T cells were cotransfected with pRL-TK plasmid and NFAT-driven IL-2 promoter luciferase reporter together with either empty vector or Ubc9 constructs. Data are represented as mean ± SEM from three experiments. (E) J14 cells were cotransfected with either empty vector, Ubc9, SLP-76, or SLP-76 plus Ubc9 constructs together with NFAT-driven IL-2 promoter luciferase reporter and pRL-TK plasmid. Data are represented as mean ± SEM from three experiments. The expressions of transfected constructs were assessed by Western blotting using Abs as indicated (lower panels). ****p < 0.0001. ns, nonsignificant.

The binding of transcription factor NFAT to IL-2 promoter is key for IL-2 transcription and production (24). We next examined the effect of Ubc9 on the binding of NFAT to IL-2 promoter in vivo by ChIP assays in mouse T cell hybridoma DC27.10 cells. DNA fragments of IL-2 promoter bound by NFAT were immunoprecipitated with an NF-ATc1–specific Ab and subjected to PCR amplification. As shown in Fig. 1B, anti-CD3 plus anti-CD28 stimulation increased NFAT binding to the IL-2 promoter. Interestingly, the amount of IL-2 promoter DNA pulled down by anti-NFATc1 Ab was significantly higher in cells overexpressing Ubc9 than empty vector control in the presence of anti-CD3 plus anti-CD28 stimulation.

Immediate tyrosine phosphorylation following TCR stimulation mediates the early activation of T cells (43). Next, we examined whether TCR-induced total tyrosine phosphorylation level is affected by overexpression of Ubc9. As shown in Fig. 1C, stimulation of T cells overexpressing Ubc9 with anti-CD3 revealed no significant difference in the total tyrosine phosphorylation as compared with an empty vector. In addition, the level of phosphorylation of specific individual molecules, including p38, ERK1/2, and AKT, was not significantly affected by overexpression of Ubc9 in response to anti-CD3 stimulation.

SLP-76 plays a key role in both TCR proximal and distal signaling steps (7). To address whether the Ubc9 potentiation of transcriptional activation of IL-2 involves SLP-76, we assessed the effect of overexpression of Ubc9 on IL-2 transcription in the SLP-76–deficient J14 Jurkat T cells. As shown in Fig. 1D, overexpression of Ubc9 in WT Jurkat cells gave rise to a 2-fold increase in NFAT-driven IL-2 promoter activity upon anti-CD3 stimulation compared with those of empty vector control. However, this enhancement effect was lost in SLP-76–deficient J14 Jurkat T cells. In keeping with this, reconstitution of SLP-76–deficient J14 cells with SLP-76 restored the Ubc9-mediated augment of IL-2 transcription in response to anti-CD3 stimulation (Fig. 1E).

Together, these data suggest that Ubc9 potentiates anti-CD3–induced IL-2 transcription by increasing NFAT binding to the IL-2 promoter, which is SLP-76 dependent.

Ubc9 interacts at the extreme C terminus of SLP-76

The SLP-76 dependence of Ubc9 potentiation of IL-2 transcription in T cells might be due to a direct interaction of Ubc9 with SLP-76 in T cells. To test this hypothesis, coimmunoprecipitation assays were performed with mouse DC27.10 hybridoma T cells by using Abs specific for SLP-76. The endogenous Ubc9 was efficiently coimmunoprecipitated with SLP-76, and anti-CD3 stimulation increased the coprecipitated Ubc9 (Fig. 2A). As a positive control, Ubc9 was readily coprecipitated by anti-RanGAP1 (44) and anti-ADAP Abs. Further, anti-CD3 ligation enhanced the amount of Ubc9 coprecipitated with SLP-76, which peaked at 5 min and declined at 10 min in DC27.10 T cells stimulated over a time course up to 10 min (Fig. 2B). These data suggest that Ubc9 binds to SLP-76 and anti-CD3 ligation promotes their association in T cells.

FIGURE 2.
FIGURE 2.

Ubc9 interacts at the extreme C- terminus of SLP-76. (A) Coimmunoprecipitation of endogenous SLP-76 and Ubc9 from resting mouse DC27.10 cells or cells stimulated by anti-CD3 for 5 min. (B) Time course of Ubc9 binding to SLP-76 upon anti-CD3 stimulation in mouse DC27.10 cells. (C) Ubc9 binds to SLP-76 in vitro. Coimmunoprecipitation analysis of SLP-76 and Ubc9 in HEK 293T cells transfected with Ubc9 and HA-tagged SLP-76 (left) or HA-tagged SLP-76–Ubc9 fusion constructs (right). (D) Ubc9 binds to extreme C terminus of SLP-76. Coimmunoprecipitation analysis of HEK 293T cells cotransfected with Ubc9 and HA-tagged SLP-76 WT or mutants. (E) Representative GST pull-down experiment using purified rGST protein, GST-tagged SLP-76-403-438, or GST-tagged SLP-76-516-533 to pull down recombinant Ubc9 protein in vitro. Pulled-down material (upper) or input (lower) were analyzed by immunoblotting using Abs against GST and Ubc9, as indicated. (F) Confocal imaging of the intracellular localization of SLP-76 (green) and Ubc9 (red) at the interface of anti-CD3–coated chamber slides (a–c) and at the cytoplasm (d–f) of Jurkat T cells transfected with EYFP–SLP-76 and mRFP-Ubc9. Scale bar, 5 μm. Results are representative of at least three independent experiments.

To determine if the interaction of Ubc9 to SLP-76 is direct, HA-tagged SLP-76 or empty vector was cotransfected with Ubc9 into HEK 293T cells where endogenous SLP-76 is absent, followed by immunoprecipitation with anti-HA Ab and immunoblotting with anti-HA and anti-Ubc9. Ubc9 was coprecipitated with HA-tagged SLP-76 (Fig. 2C, left panel), whereas HA-tagged RanGAP1 and ADAP served as positive controls, and SKAP1 was the negative control for Ubc9 binding (33). Further, Ubc9 was readily coprecipitated with a fusion version of SLP-76 with a Ubc9 attached to its C terminus (Fig. 2C, right panel).

To identify the site/region in SLP-76 that is responsible for Ubc9 binding, various HA-tagged SLP-76 truncation or deletion mutants were coexpressed with Ubc9 in HEK 293T cells. As shown in Fig. 2D, whereas SLP-76 mutants that harbor deletion at the N terminus (aa 1–201) as well as various truncations at aa 201–516 exhibited Ubc9 binding in a level comparable to those of WT, a mutant with a deletion of 17 aa at the extreme C terminus (aa 516–533) failed to bind to Ubc9. Comparable expressions of HA-tagged SLP-76 WT and mutants were confirmed by anti-HA blotting (lower panel). To further verify whether SLP-76 binding to Ubc9 is direct via its extreme C terminus, GST-tagged SLP-76 (aa 516–533) was expressed and purified from E. coli and mixed with the recombinant Ubc9 protein, followed by an in vitro GST pull-down assay. As shown in Fig. 2E, Ubc9 was readily pulled down by GST-SLP-76-516-533, but not by the GST protein alone or a control GST fusion protein containing residues 403–438 of SLP-76, confirming the interaction between SLP-76 and Ubc9 is indeed direct.

Further, SLP-76 is assembled into microclusters at the sites of TCR engagement (45). To assess whether Ubc9 could colocalize with SLP-76 microclusters upon anti-CD3 stimulation, we cotransfected the Jurkat T cells with EYFP–SLP-76 and mRFP-Ubc9 and placed the cells on anti-CD3–coated glass chambered slides. The confocal microscopy images showed that EYFP–SLP-76 (green) formed microclusters at the interface between transfected cells and anti-CD3–coated slides (Fig. 2Fa). Although the majority of Ubc9 was detected in the cytoplasm (Fig. 2Fe), a small portion of mRFP-Ubc9 (red) also displayed a pattern of small fluorescent puncta at the stimulatory interface (Fig. 2Fb). Interestingly, the mRFP-Ubc9 showed a substantial degree of colocalization with SLP-76 both at the cytoplasm and at the stimulatory interface. Moreover, the colocalization was also found for a portion of endogenous Ubc9 and SLP-76 proteins at the cytoplasm, which was further increased in response to PMA plus ionomycin stimulation in Jurkat T cells (Supplemental Fig. 1).

Collectively, these data indicate that Ubc9 directly interacts with SLP-76 and the region spanning aa 516–533 at the extreme C terminus of SLP-76 is required for Ubc9 binding.

SLP-76 is SUMOylated at residues Lys266 and Lys284, and anti-CD3 stimulation enhances SLP-76 SUMOylation

Given that SLP-76 binds to Ubc9, we next examined whether SLP-76 is a target of Ubc9-mediated SUMOylation. When immunoprecipitation assay was performed with anti–SLP-76 Ab and immunoblotted with anti–SLP-76 Ab in mouse hybridoma DC27.10 T cells with anti-CD3 stimulation over a time course of 45 min, two supershifted bands with molecular masses of ∼120 and 130 kDa were consistently observed in addition to the major 76-kDa SLP-76. The membrane was then stripped and reprobed with anti-SUMO1, which detected the same two supershifted bands, suggesting the supershifted bands correspond to SUMOylated SLP-76. Moreover, the band intensities were increased after a 30-min stimulation and further increased after prolonged stimulation for 45 min (Fig. 3A, left panel). This anti-CD3–enhanced SLP-76 SUMOylation was also assessed in human PBLs. The two supershifted bands were readily detected in unstimulated cells, whereas anti-CD3 stimulation increased the intensity of these supershifted bands (Fig. 3A, right panel). These data suggest that SLP-76 is SUMO modified in T cells and anti-CD3 stimulation enhances SLP-76 SUMOylation.

FIGURE 3.
FIGURE 3.

SLP-76 is SUMOylated at residues Lys266 and Lys284, and anti-CD3 stimulation enhances SLP-76 SUMOylation. (A) In vivo SUMO modification of SLP-76. Left panel, DC27.10 cells stimulated with anti-CD3 for 0–45 min were subjected to immunoprecipitation with anti–SLP-76 Ab and immunoblotting using anti–SLP-76 and anti-SUMO1 Abs. Right panel, Human PBLs stimulated with or without anti-CD3 for 45 min were lysed and subjected to immunoprecipitation with anti–SLP-76 Ab followed by immunoblotting using anti–SLP-76. (B) SUMO modification of SLP-76 by Ubc9 fusion–directed SUMOylation method. HEK 293T cells were cotransfected with HA-tagged SLP-76 WT–Ubc9 fusion or SLP-76 mutants–Ubc9 fusion with FLAG-tagged SUMO1 plasmid. Immunoprecipitation was performed with anti-HA Ab, followed by Western blotting with anti-Ubc9 and anti–FLAG-tag, as indicated. (C) SUMOylation-site identification by LC-MS/MS analysis. HEK 293T cells were cotransfected with FLAG-tagged SUMO1 WT or SUMO1-T95R and HA-tagged SLP-76Δ438-516-Ubc9 fusion plasmid. The immunoprecipitates by anti-HA were subjected to Western blotting as in (B) (upper left) and Coomassie Brilliant Blue staining (upper right). The gel band (red rectangle) was analyzed by LC-MS/MS. MS spectra and ion assignment of the identified SUMOylated peptides of SLP-76 (lower). (D) Upper panel, Schematic representation of the Ubc9-interacting region in the extreme C terminus and the two identified SUMOylation sites K266 and K284 within the central PRR of SLP-76. Lower panel, Alignments of SLP-76 sequences from different species surrounding the SUMOylation sites Lys 266 and Lys 284 (highlighted in red). (E) Point mutation at K266 and K284 failed to facilitate SLP-76 SUMOylation. Left panel, The HA-tagged SLP-76 WT–Ubc9 or SLP-76 mutant–Ubc9 fusions and FLAG-tagged SUMO1 were transfected into SLP-76–deficient J14 T cells. Cell extracts were immunoprecipitated by anti-HA, followed by immunoblotting with anti-HA. Right panel, Same amount of stable J14 cells expressing SLP-76 WT, K266R, and K284R constructs were stimulated with anti-CD3 for 45 min, followed by immunoprecipitation with anti–SLP-76 Ab and immunoblotting with anti–SLP-76 and anti-SUMO1 Abs. Results are representative of at least three independent experiments.

To further verify SLP-76 SUMOylation and determine the SUMOylation sites, we used a Ubc9 fusion-directed SUMOylation method, which allows efficient in vitro SUMOylation of proteins (46). HEK 293T cells were transfected with HA-tagged SLP-76–Ubc9 fusion construct alone or together with FLAG-tagged SUMO1 construct. The expressed HA-tagged SLP-76–Ubc9 fusion protein was immunoprecipitated with anti-HA Ab, followed by immunoblotting with anti-Ubc9 and anti-FLAG. As shown in Fig. 3B, coexpression of SLP-76–Ubc9 fusion with SUMO1 revealed one major band at 100 kDa and a few bands at 120–150 kDa, with lower mobility against unsumoylated SLP-76–Ubc9 (lane 2). These supershifted bands reacted with anti-FLAG Ab, suggesting these bands are SUMOylated versions of SLP-76–Ubc9. Interestingly, when coexpressing a truncation mutant at aa 438–516 of SLP-76–Ubc9 (hereafter referred to as SLP-76Δ438-516-Ubc9) with SUMO1, the intensity of the supershifted bands was significantly enhanced (lane 4 versus lane 2), suggesting that the region spanning aa 438–516 is inhibitory for the SUMOylation of SLP-76. That the supershifted bands were caused by SUMOylation was further confirmed by the intensity of the supershifted bands being significantly decreased upon coexpression of the FLAG-tagged sentrin-specific protease 1 (SENP1) (lane 5 versus lane 4), a SUMO-specific protease that removes SUMO moiety from substrates (28). Moreover, when coexpressing the Ubc9 binding–deficient truncation mutant at aa 516–533 of SLP-76Δ438-516-Ubc9 (SLP-76Δ438-533-Ubc9) with SUMO1, the intensities of the supershifted bands were also decreased (lane 6 versus lane 4), suggesting that Ubc9 binding to SLP-76 is required for the maximal SLP-76 SUMOylation level.

We thus used this truncation mutant SLP-76Δ438-516 for the mapping of the SUMOylation sites within SLP-76 in vitro by LC-MS/MS analysis. Digestion of SUMO1-T95R–conjugating proteins will generate a signature peptide showing an increase of 114.1 Da in peptide mass due to the covalently attached diglycine tag to the modified lysine, which can be easily analyzed by mass spectrometry (47). We thus used a T95R mutant of SUMO1 (SUMO1-T95R) instead of WT SUMO1 in the in vitro SUMOylation assay prior to LC-MS/MS analysis. To assess if the SUMO1-T95R can also be efficiently attached to SLP-76 for SUMOylation, HEK 293T cells were cotransfected with SLP-76Δ438-516-Ubc9 alone or in combination with SUMO1 WT or SUMO1-T95R. Consistent with SUMO1 WT, coexpression with SUMO1-T95R also caused the SUMOylation of SLP-76Δ438-516-Ubc9 as shown by multiple supershifted bands recognized by both anti-Ubc9 and anti-FLAG Abs, albeit the molecular mass was slightly smaller than that in coexpression with SUMO1 WT (Fig. 3C, upper left panel). The gel band corresponding to the supershifted bands was excised from Coomassie stained gel and subjected to trypsin digestion, followed by LC-MS/MS analysis (Fig. 3C, upper right panel). We found that lysine residues at positions 266 and 284 in SLP-76 harbored a diglycine tag with a 114.1-Da increase in peptide mass (Fig. 3C, lower panel). Sequence alignment of SLP-76 from different species showed that Lys266 and Lys284 and their surrounding sequences are conserved (Fig. 3D).

Next, we substituted these lysine residues individually with arginine on SLP-76Δ438-516-Ubc9 fusion construct and transfected into SLP-76–deficient J14 T cells with FLAG-tagged SUMO1. The in vitro SUMOylation assay showed that whereas coexpression of SUMO1 significantly enhanced the SUMOylation level of SLP-76Δ438-516-Ubc9 fusion (Fig. 3E, left panel), the SUMOylation levels of SLP-76 K266R-Ubc9, K284R-Ubc9, and dual mutant K266/284R-Ubc9 fusions were decreased. These data suggest that Lys266 and Lys284 were the two major SUMOylation sites within SLP-76. Similar results were obtained from in vivo SUMOylation assay. Full-length SLP-76 WT or K266R and K284R mutants were stably reconstituted into J14 cells, followed by stimulation with anti-CD3 for 45 min. Consistent with Fig. 3A, a supershifted band at ∼110 kDa was consistently observed in addition to the 76-kDa SLP-76, which was also recognized by anti-SUMO1 Ab (Fig. 3E, right panel); however, the supershifted band at ∼125 kDa disappeared, most likely due to the less-efficient SUMOylation machinery with reconstituted HA-tagged SLP-76 in J14 cells. In contrast, again, in the stable SLP-76 K266R- and SLP-76 K284R-reconstituted J14 cells, no such supershifted band was observed. These data suggest that SLP-76 is SUMOylated at two conserved residues, Lys 266 and Lys 284, within SLP-76.

SLP-76 SUMOylation is not required for TCR proximal signaling

We next examined the role played by SLP-76 SUMOylation in TCR proximal signaling events. First, we compared the microcluster formation of Jurkat T cells expressing EYFP-tagged SLP-76 WT and SLP-76 SUMO-site mutants K266R and K284R or K266/284R. As shown in Fig. 4A, SLP-76 SUMO-site mutants K266R, K284R, and K266/284R supported formation of SLP-76 microclusters in response to anti-CD3 stimulation. The number of clusters per cell and the average size of cluster in the cells expressing K266R, K284R, or dual K266/284R were similar as compared with cells expressing SLP-76 WT.

FIGURE 4.
FIGURE 4.

SLP-76 SUMOylation is not required for TCR proximal signaling. (A) Confocal imaging of EYFP–SLP-76 WT, SLP-76 K266R, and SLP-76 K284R microclusters in Jurkat cells on anti-CD3–coated chamber slides. Scale bar, 5 μm. Histograms showed the number (left) and size (right) of the microclusters. Data are represented as mean ± SEM from 30 to 40 cells from three experiments. (B) J14 cells expressing EYFP–SLP-76 WT, K266R, K284R, or K266/284R mutants were either left resting or stimulated with anti-CD3 for 5 min, followed by immunoprecipitation with anti-GFP and immunoblotting with Abs against phosphotyrosine (4G10), GADS, PLC-γ1, or GFP-tag (left). Histogram showing the ratio values of band intensity as quantified by densitometry and normalized to the results obtained in resting cells expressing SLP-76 WT in the same experiment (right). Data are represented as mean ± SEM from four experiments. (C) Confocal imaging of the intracellular localization of SLP-76 WT, K266R, K284R, K266/284R (green), and Ubc9 (red) at the interface of anti-CD3–coated chamber slides with Jurkat T cells transfected with EYFP-tagged SLP-76 mutants and mRFP-Ubc9 (left). Quantification of the Pearson correlation coefficient values of the SLP-76 and Ubc9 in each condition (right); n = 20–25 images per condition from three experiments. Scale bar, 5 μm.

We also assessed whether the tyrosine phosphorylation of SLP-76 is affected by expressions of SLP-76 SUMO-site mutants upon TCR stimulation. Consistent with the lack of an effect on microcluster formation, Jurkat T cells expressing SLP-76 WT, K266R, K284R, or dual K266/284R responded effectively and retained a similar level of increase in SLP-76 tyrosine phosphorylation in response to anti-CD3 stimulation (Fig. 4B). The SUMOylation sites at Lys266 and Lys284 locate within the central PRR of SLP-76, a region responsible for GADS and PLCγ1 binding (11, 48). SLP-76 K266R, K284R, and dual K266/284R also coprecipitated with both GADS and PLCγ1, and the bindings were strengthened upon anti-CD3 stimulation to a comparable level as relative to those of WT SLP-76. Further, as shown in Fig. 4C, the microclusters formed by the SLP-76 SUMO-site mutants K266R, K284R, and dual K266/284R colocalized with Ubc9 puncta at the interface of the transfected Jurkat T cells and anti-CD3–stimulatory surface.

Together, these data suggest that SUMOylation of SLP-76 would not affect the TCR-triggered SLP-76–mediated proximal signal initiation.

Ubc9 synergizes with SLP-76 to augment the TCR-mediated IL-2 transcription in an SLP-76 SUMOylation-dependent manner

Next, we assessed whether Ubc9 cooperates with SLP-76 to drive the TCR-mediated induction of IL-2 transcription. Overexpression of SLP-76 resulted in a 3.6-fold increase in the NFAT-driven IL-2 promoter activity in response to CD3 stimulation as compared with those of empty vectors (Fig. 5A, left panel). Interestingly, overexpression of SLP-76 together with Ubc9 gave rise to a further 6-fold increase as compared with that of overexpression of SLP-76 alone, suggesting there is a synergy between SLP-76 and Ubc9 in the TCR-induced IL-2 transcription. In contrast, Ubc9 failed to collaborate with ADAP or SKAP1 in the anti-CD3–induced NFAT-driven IL-2 promoter activity. The synergy between SLP-76 and Ubc9 in the induction of TCR-mediated IL-2 transcription was further confirmed by quantitative PCR analysis. Whereas overexpression of SLP-76 resulted in a 1.5-fold increase in IL-2 mRNA level in response to anti-CD3 stimulation, coexpression of SLP-76 and Ubc9 augmented the increase to 3-fold relevant to that of empty vectors (Fig. 5A, right panel). However, Ubc9 and SLP-76 synergy was not observed on NF-κB–driven IL-2 promoter activity (Fig. 5B). Further, Ubc9 retained the synergy with SLP-76 on anti-CD3–induced IL-2 promoter activity in stable ADAP-knockdown Jurkat T cells (Fig. 5C). These data suggest that Ubc9 synergizes with SLP-76 selectively for NFAT but not NF-κB transcription in T cell activation.

FIGURE 5.
FIGURE 5.

Ubc9 synergizes with SLP-76 to augment the TCR-mediated IL-2 transcription in an SLP-76 SUMOylation-dependent manner. (A) Ubc9 synergizes with SLP-76 but not ADAP or SKAP1 in promoting IL-2 transcription. Jurkat T cells were transfected with vector, SLP-76, ADAP, and SKAP1 alone or in combination with Ubc9 constructs plus an NFAT-driven IL-2 promoter luciferase reporter and pRL-TK plasmid (left). IL-2 mRNA level of mouse DC27.10 T cells transfected with vector, Ubc9, SLP-76, or SLP-76 plus Ubc9 constructs followed by anti-CD3 stimulation for 6 h (right). Data are represented as mean ± SEM from four experiments. (B) Jurkat T cells were cotransfected with vector, Ubc9, SLP-76, or SLP-76 plus Ubc9 constructs together with NF-κB–driven IL-2 promoter luciferase reporter and pRL-TK plasmid. Data are represented as mean ± SEM from four independent experiments. (C) Knockdown of ADAP did not affect the Ubc9–SLP-76 synergy on IL-2 transcription. Data are represented as mean ± SEM from three experiments. (D) SLP-76 SUMO-site mutants failed to support SLP-76–Ubc9 synergy on the anti-CD3–induced IL-2 transcription. Left, Jurkat T cells were transfected with SLP-76 WT, K266R, K284R, K266/284R, or d516-533 mutants alone or in combination with Ubc9 constructs plus an NFAT-driven IL-2 promoter luciferase reporter and pRL-TK plasmid. Data are represented as mean ± SEM from four independent experiments. Right, ELISA of IL-2 in the supernatant of mouse splenocytes expressing SLP-76 WT, K266R, K284R, or K266/284R alone or in combination with Ubc9 constructs. Data are represented as mean ± SEM from three independent experiments. (E) Dominant negative Ubc9-C93S impaired the anti-CD3–induced NFAT promoter activity. Jurkat T cells were transfected with vector, SLP-76 WT, or in combination with Ubc9 or Ubc9 C93S plus an NFAT-driven IL-2 promoter luciferase reporter and pRL-TK plasmid. Data are represented as mean ± SEM from four independent experiments. The expressions of transfected constructs were assessed by Western blotting using Abs, as indicated (lower or right panels). *p < 0.05, ****p < 0.0001. ns, nonsignificant.

To address whether SUMOylation is required for SLP-76 synergy with Ubc9 in the induction of TCR-mediated IL-2 transcription, SLP-76 WT or SLP-76 SUMO-site mutants were transfected alone or in combination with Ubc9 in T cells. As shown in Fig. 5D, left panel, overexpression of either SLP-76 K266R, K284R, or dual K266/284R individually in Jurkat T cells resulted in a significant increase in NFAT-driven IL-2 promoter activity upon anti-CD3 stimulation, which reached a comparable level as overexpressed WT SLP-76. However, it failed to support the synergy with Ubc9 in augmentation of NFAT-driven IL-2 promoter activity, as shown by an ∼40–60% decrease compared with those of WT SLP-76 with Ubc9. Moreover, the Ubc9 binding–deficient SLP-76 d516-533 mutant also completely lost the ability to potentiate the anti-CD3–mediated IL-2 promoter activity, resulting in a much lower IL-2 promoter activity (34% of the SLP-76 WT). Furthermore, the coexpression of Ubc9 with SLP-76 K266R, K284R, and K266/284R showed a similar level of increase in IL-2 production relative to those of SLP-76 K266R, K284R, and K266/284R alone (Fig. 5D, right panel).

Last, to determine whether the SLP-76–Ubc9 synergism requires the E2 ligase activity of Ubc9 for the induction of IL-2 transcription, we generated a Ubc9-dominant negative mutant (C93S), which is deficient in SUMO1 conjugation (49). When Ubc9 C93S was overexpressed in Jurkat T cells, the sharp increase of NFAT-driven IL-2 promoter activity potentiated by WT Ubc9 was reduced to the basal levels (Fig. 5E). Moreover, the synergistic effect showed in the coexpression of SLP-76 with WT Ubc9 disappeared, as well. These results suggest that Ubc9 and its synergism with SLP-76 in the augmentation of TCR-mediated IL-2 transcription are SLP-76 SUMOylation dependent.

SLP-76 SUMOylation is required for TCR-induced assembly of Ubc9-NFAT complex

Binding of NFAT to the promoter of IL-2 is essential for the inducible transactivation of IL-2 promoter in activated T cells (21). Ubc9 interacts with NFAT and SUMOylates NFAT, which prolongs the stay of NFAT in nucleus, thus activating the NFAT transcriptional activity (34, 35). Next, we assessed the role of SLP-76–Ubc9 interaction in the TCR-induced assembly of the Ubc9-NFAT complex by coimmunoprecipitation assays. In both Jurkat T cells and mouse DC27.10 T cells, as expected, Ubc9 was effectively coprecipitated with NFATc1, and the binding was further increased following anti-CD3 stimulation (Fig. 6A). In contrast, in the SLP-76–deficient J14 cell, only a low level of Ubc9 coprecipitated by anti-NFATc1 from both resting and anti-CD3–stimulated cells, suggesting that SLP-76 is required for Ubc9-NFAT association in T cells. Further, the formation of the Ubc9-NFAT complex was restored by stable reconstitution of SLP-76–deficient J14 cells with WT SLP-76 but not with the SUMO-site mutants K266R or K284R (Fig. 6B).

FIGURE 6.
FIGURE 6.

SLP-76 SUMOylation is required for TCR-induced assembly of Ubc9-NFAT complex. (A) Mouse DC27.10 T cells, Jurkat T cells, or J14 cells were stimulated with anti-CD3 for 60 min or left unstimulated. Immunoprecipitation was performed with anti-NFATc1 Ab, followed by immunoblotting with the indicated Abs. (B) J14 cells stably reconstituted with SLP-76 WT, or mutants were stimulated with anti-CD3 for 60 min. Immunoprecipitation was performed as in (A). (C) The HA-tagged SLP-76 WT or SLP-76 SUMO-site mutants were transfected into HEK 293T cells together with FLAG-tagged NFATc1 and Ubc9 plasmid. Immunoprecipitation was performed with anti-FLAG Ab followed by immunoblotting using anti-FLAG and anti-Ubc9. (D) EMSA of nuclear fractions from mouse DC27.10 T cells transfected with SLP-76 WT or mutants and stimulated with anti-CD3/CD28 for 1 h. Probes containing WT or a mutated NFAT-binding site at the −90 and −45 from IL-2 promoter were Cy3 labeled. Cold probes were unlabeled oligos with the same sequence as WT probes. Results are representative of at least three independent experiments.

Similar to results obtained with J14 cells, in HEK 293T cells cotransfected with FLAG-NFATc1 and Ubc9 together with empty vector, SLP-76 WT, or SLP-76 mutants, the amount of Ubc9 coprecipitated with NFATc1 was significantly higher upon coexpression of SLP-76 WT as compared with the vector control (Fig. 6C). By contrast, the SUMO-site SLP-76 mutants K266R, K284R, or K266/K284R and the Ubc9 binding–deficient SLP-76 mutant (SLP-76Δ516-533) failed to do so. Moreover, EMSA with two Cy3-labeled oligonucleotide probes from the IL-2 promoter centered at the −90 and −45 nt NFAT binding site (42), respectively, showed that the complex formation between NFAT and IL-2 promoter probe was significantly reduced in the analysis with the nuclear extract from cells expressing SLP-76 SUMO-site mutant K266/K284R and Ubc9 binding–deficient SLP-76Δ516-533 mutant than SLP-76 WT (Fig. 6D). The protein–DNA interaction in the shifted complexes was specific because it was significantly decreased when mutant probes bearing mutations made within the consensus NFAT binding site were used (Fig. 6D, lanes 15 and 16) or when an excessive amount of unlabeled probes was included (Fig. 6D, lanes 17 and 18). Taken together, these data suggest that SLP-76 binding to Ubc9 and its SUMOylation are required for the TCR-induced association of NFAT with Ubc9 in T cells.

Discussion

SUMOylation has been implicated in the regulation of diverse cellular processes (26). However, our current understanding of the role of SUMOylation in TCR signaling and T cell activation is that it is still largely unknown. We previously reported the binding of the adaptor protein SLP-76 to the SUMOylated version of RanGAP1 of the nuclear pore complex and demonstrated a role for the interaction in the transport of NFATc1 into the nucleus of T cells (25). In this context, the E2 conjugase, Ubc9, mediates the transfer of SUMO protein to the target substrates. In this study, we extend our observations by showing 1) Ubc9 binds directly to SLP-76, the key TCR adaptor protein, at the extreme of C terminus spanning residues 516–533, and the binding is enhanced upon anti-CD3 stimulation; 2) SLP-76 is targeted by SUMO in a Ubc9-dependent manner in vivo and in vitro, and the SUMOylation sites were mapped at two conserved lysine residues, K266 and K284, within the PRR; 3) Ubc9 synergizes with SLP-76 to potentiate the TCR-induced IL-2 transcription, which requires SLP-76 SUMOylation; and 4) although not required for proximal signaling in T cell activation, SLP-76 SUMOylation is required for the TCR-induced assembly of functional complex Ubc9-NFAT required for IL-2 transcription. Our study is, to our knowledge, the first to demonstrate SUMOylation of immune adaptor SLP-76 and the mediation of this event by an interaction with Ubc9 in T cells.

SUMOylation commonly takes place on lysine residues of the canonical consensus motif ψKxE (where ψ is a hydrophobic residue and x represents any residue) recognized by Ubc9 (50). However, a number of SUMO substrates are modified on sites that do not conform to this consensus motif (51, 52). Despite the absence of canonical motifs, we found that SLP-76 was SUMOylated in vitro and in vivo in a manner dependent on the interaction between SLP-76 and Ubc9. The identified SUMOylation sites K266 and K284 within the central PRR of SLP-76 do not follow this canonical motif. Thus, SLP-76 constitutes an unusual SUMO substrate of which the Ubc9-interacting region is separate from the SUMOylation sites. Similar examples are also found in other SUMO substrates, such as nuclear receptor corepressor (N-CoR) and adenosine diphosphate ribosylation factor-like protein 13 (ARL-13), in which the SUMOylation sites and Ubc9-interacting sites are not overlapped (53, 54). Further, deletion of the residues 438–516 on SLP-76 resulted in a marked elevation in the level of SUMOylation, suggesting this stretch of amino acids on SLP-76 may mediate intramolecular interaction that blunts the detection of SUMOylation as reported (55–57). So far, researchers have identified more than 300 substrates of the SUMO pathway that can be classified into different categories, including transcription factors, nucleoporins, viral proteins, kinases, etcetera (58). The identification of SLP-76 as a SUMO substrate expands the repertoire of SUMOylation targets, which is the first example of a T cell immune adaptor that is SUMO modified by interaction with Ubc9.

Further, we found SLP-76 SUMOylation had played functions in the late events of TCR activation. Although having no effect on anti-CD3–induced tyrosine phosphorylation of substrates in T cells, Ubc9 overexpression significantly potentiated the TCR-mediated IL-2 gene transcription and production, a classic indicator of T cell activation, in an SLP-76–dependent manner. Accordingly, the Ubc9-mediated SUMOylation of SLP-76 was dispensable for proximal signaling events, such as SLP-76 tyrosine phosphorylation, association with PLCγ1 and GADS, and microcluster formation. These observations are in agreement with the results showing that TCR proximal signaling was unaffected in Ubc9 knockdown cells (33) and KO primary T cells (32). Interestingly, it was recently reported that TCR-induced SUMOylation of PLCγ1 K54 promotes its association with SLP-76 and GADS (30). One possible explanation for this discrepancy is that in contrast with our observation that SUMOylation of SLP-76 had no effect on its microcluster formation in response to TCR stimulation, TCR-induced SUMOylation of PLCγ1 promotes its assembly of microclusters (30), which could facilitate PLCγ1 association with the clusters of SLP-76 and GADS, as does the dynamic interaction between SLP-76 and LAT microclusters at T cell membrane (45). Ubc9–SLP-76 functional collaboration was further supported by the observation that Ubc9 synergized with SLP-76 to augment IL-2 gene transcription in vivo and in vitro. The enzymatically inactive Ubc9 mutant C93S failed to do so. In addition, there was no synergy between Ubc9 and the SLP-76 SUMO-site mutant K266/284R. Furthermore, Ubc9 binding–deficient mutant SLP-76Δ516-533 also lost an ability to synergize with Ubc9. These results suggest that Ubc9–SLP-76 functional collaboration in T cell activation depends on both their direct interaction and the SUMOylation of SLP-76. We have previously showed that Ubc9 also interacts with SLP-76–associated ADAP for regulation of T cell adhesion (33). However, despite its interaction with Ubc9, ADAP is not a substrate for SUMO conjugation (33). Moreover, Ubc9 and SLP-76 synergizes to potentiate the IL-2 transcription in a SUMOylation-dependent manner, whereas the Ubc9-ADAP module has no apparent effect on IL-2 transcription but selectively regulates T cell adhesion in a SUMOylation-independent manner. Thus, by interplaying with different immune adaptors, Ubc9 regulates discrete T cell functions.

Ubc9 forms a functional complex with NFAT and mediates NFAT SUMOylation, which regulates transcription activities of NFAT by prolonging the stay of NFAT in nucleus (34, 35). In line with the finding that the nuclear translocation of NFATc1 is impaired in thymocytes from Ubc9-deficient mice (32), our in vivo ChIP assay showed that Ubc9 substantially promoted NFAT binding to the IL-2 promoter in T cells, suggesting that the expression of IL-2 may depend on Ubc9-NFAT complex enrichment at the IL-2 promoter. Despite TCR-induced association and SUMOylation of SLP-76 by Ubc9, SLP-76 SUMOylation is not required for TCR proximal signaling. Rather, both SLP-76 and its SUMOylation are required for the assembly of Ubc9-NFAT complex and the binding of NFAT to IL-2 promoter and subsequent downstream distal TCR-mediated IL-2 gene transcription and production. Based on the current results, we proposed that Ubc9 interacts with and SUMOylates SLP-76, which is required for the recruitment of Ubc9 to the vicinity of NFAT and the assembly of the functional Ubc9-NFAT complex for IL-2 transcription in T cell activation. Ubc9–SLP-76 synergy in potentiating TCR-mediated IL-2 transcription and production could be achieved in several ways: first, TCR-induced SLP-76 interaction with Ubc9 and its SUMOylation can recruit Ubc9 to the vicinity of NFAT, promoting the assembly of the Ubc9-NFAT complex; second, Ubc9-mediated SLP-76 SUMOylation may provide an immediate docking site that facilitates the entry of Ubc9-associated NFAT binding to the IL-2 promoter; and third, SLP-76 SUMOylation could increase the interaction with SUMO-RanGap1 at nuclear pore complex, which is essential for NFAT nuclear translocation for activation of IL-2 promoter (25). However, the question that still remains unsolved is how SUMOylated SLP-76 is able to affect the assembly of the Ubc9-NFAT complex.

Our findings thus provide a new (to our knowledge) insight that the Ubc9–SLP-76 module acts as a novel regulatory layer on the controls of T cell activation. Ubc9 exerts functionally separable effects on T cell activation and adhesion, dependent on its ability to bind specifically to immune adaptor SLP-76 or ADAP. These findings shed new light on the ability of the SUMO pathway to influence T cell activation.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Feng Ma (Suzhou Institute of System Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College) for providing equipment on nucleofection.

Footnotes

  • This work was supported by grants from the National Natural Science Foundation of China (31470840 to H.L.) and the Suzhou Key Program Special Fund (KSF-E-30 to H.L.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ADAP
    adhesion and degranulation-promoting adapter protein
    ChIP
    chromatin immunoprecipitation
    GADS
    Grb2-related adaptor downstream of Shc
    HA
    hemagglutinin
    LC-MS/MS
    liquid chromatography–tandem mass spectrometry
    PLCγ1
    phospholipase Cγ1
    PRR
    proline-rich region
    RanGAP1
    Ran GTPase activating protein 1
    SKAP1
    src kinase associated phosphoprotein 1
    SLP-76
    SH2 domain containing leukocyte phosphoprotein of 76 kDa
    SUMO
    small ubiquitin-like modifier
    WT
    wild type.

  • Received May 14, 2019.
  • Accepted September 30, 2019.

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