TCR signaling promotes the assembly of RanBP2/RanGAP1-SUMO1/Ubc9 nuclear pore subcomplex via PKC-θ-mediated phosphorylation of RanGAP1

The nuclear pore complex (NPC) is the sole and selective gateway for nuclear transport and its dysfunction has been associated with many diseases. The NPC subcomplex RanBP2, which consists of RanBP2 (Nup358), RanGAP1-SUMO1 and Ubc9, regulates the assembly and function of the NPC. The roles of immune signaling in NPC assembly remain poorly understood. Here, we show that, following TCR stimulation, protein kinase C-θ (PKC-θ) directly phosphorylates RanGAP1 to facilitate RanBP2 subcomplex assembly and nuclear import and, thus, the nuclear translocation of AP-1 transcription factor. Mechanistically, TCR stimulation induces the translocation of activated PKC-θ to the NPC, where it interacts with and phosphorylates RanGAP1 on Ser504 and Ser506. RanGAP1 phosphorylation increases its binding affinity for Ubc9, thereby promoting sumoylation of RanGAP1 and, finally, assembly of the RanBP2 subcomplex. Our findings reveal an unexpected role of PKC-θ as a direct regulator of nuclear import and uncover a phosphorylation-dependent sumoylation of RanGAP1, delineating a novel link between TCR signaling and assembly of the RanBP2 NPC subcomplex.


PKC-θ deficiency inhibits the association of RanGAP1 with the NPC by reducing its sumoylation 197
Given the association between PKC-θ and RanGAP1, we next explored the physiological significance of this 198 interaction. We first inspected the localization of RanGAP1 in resting T cells from wild type or Prkcq −/− mice.          . We therefore hypothesized that PKC-θ-mediated phosphorylation of RanGAP1 might regulate the 309 interaction between Ubc9 and RanGAP1 and, furthermore, that RanGAP1 phosphorylation would be required 310 for assembly of this subcomplex. Hence, we first examined whether RanGAP1 sumoylation affects its binding 311 to Ubc9 and found that a non-sumoylated RanGAP1 mutant (K524R) was still capable of associating with Ubc9 312 ( Figure 5A). Next, we generated two RanGAP1 mutants: One, which was mutated at both its sumoylation 313 (K524R) and phosphorylation (S504A/S506A) sites (HA-RanGAP1 AA /K524R), and another K524R mutant 314 with a replacement of Ser 504 and Ser 506 PKC-θ phosphorylation sites by a glutamic acid as a phosphorylation 315 mimic (HA-RanGAP1 EE /K524R). We then analyzed the association of these mutants with Ubc9 by reciprocal 316 co-IP and found that, compared with HA-RanGAP1-K524R, HA-RanGAP1 AA -K524R showed a decreased 317 association with Ubc9, while HA-RanGAP1 EE -K524R had a stronger interaction ( Figure 5A,B). This result 318 indicates that RanGAP1 phosphorylation promotes its binding to Ubc9, and provides an explanation for our 319 finding that mutation of the RanGAP1 PKC-θ phosphorylation sites inhibits its sumoylation ( Figure 4F). 320 Next, we transfected Jurkat T cells with HA-RanGAP1, HA-RanGAP1 AA , or HA-RanGAP1 EE and analyzed 321 their association with Ubc9 and RanBP2 by reciprocal co-IP. HA-RanGAP1 AA bound to Ubc9 and RanBP2 less 322 effectively than non-mutated HA-RanGAP1; in contrast, HA-RanGAP1 EE bound more effectively to Ubc9 and 323 RanBP2 ( Figure 5C,D). As expected, due to its stronger association with Ubc9, the RanGAP1 EE mutant protein 324 displayed an increased ratio of RanGAP1-SUMO1 to RanGAP1 relative to the two other RanGAP1 proteins 325 ( Figure 5E). Similar result was also observed when HA-RanGAP1 EE transfected cell lysates was blotted with 326 RanGAP1 antibody instead of HA antibody (Figure 5─figure supplement 1A). These results reveal that PKC-327 θ-mediated phosphorylation of RanGAP1 is essential for assembly of the RanBP2/RanGAP1-SUMO1/Ubc9 328 subcomplex in NPCs. Intriguingly, in silico analysis showed that replacement of the two PKC-θ phosphorylation 329 sites in RanGAP1 by glutamic acid (S504E/S506E) increased the overall structural stability of RanGAP1 RanGAP1 to increase its association with Ubc9 and, in turn, the sumoylation of RanGAP1, thereby facilitating 333 assembly of the RanBP2/RanGAP1-SUMO1/Ubc9 subcomplex upon TCR stimulation.

339
The ratio of RanGAP1-SUMO1 to RanGAP1 in the WCL of (C) and (D) is quantified in (E), quantification is based on three biological 340 replicates. *P < 0.05 (one-way ANOVA with post hoc test). Data are representative of three biological replicates. Based on the results above (Figure 4 and Figure 5), we hypothesized that the non-phosphorylatable RanGAP1 346 mutant (RanGAP1 AA ) will have impaired ability to promote nuclear transport of key TCR-activated 347 transcription factors that are required for productive T cell activation. As a positive control, we generated a confirmed that expression of this mutant resulted in blocked TCR-induced nuclear translocation of NFATc1, 351 p65 (NF-κB), and c-Jun and c-Fos (AP-1) (Figure 6─figure supplement 1C,D). 352 We next determined whether transfection of the RanGAP1-KD cell line with wild-type RanGAP1 or 353 RanGAP1 AA can rescue the nuclear translocation of NFATc1, p65, or AP-1, with unedited Jurkat cells serving 354 as a negative control. Using subcellular fractionation ( Figure 6A) or confocal microscopy ( Figure 6B,C), we 355 observed that the defective nuclear translocation of these transcription factors in RanGAP1-KD cells was largely 356 rescued by wild-type RanGAP1, but not by the RanGAP1 AA mutant. Similarly, while stimulated RanGAP1-KD 357 cells displayed reduced IL-2 production, which is known to require the cooperative activity of the above 358 transcription factors, as compared to control cells, transfection with wild-type RanGAP1 rescued IL-2 359 production, while RanGAP1 AA did not ( Figure 6D    Here, we have demonstrated that upon TCR plus CD28 costimulation, PKC-θ phosphorylates RanGAP1 to 387 promote its interaction with Ubc9 and increase the sumoylation of RanGAP1, which, in turn, enhances assembly 388 of the RanBP2 subcomplex and, thus, directly promotes the nuclear import of AP-1, NFAT and NF-κB ( Figure  389   7). Thus, our work reveals a novel signaling axis, TCR-PKC-θ-RanGAP1, which regulates T-cell activation via 390 control of nucleo-cytoplasmic transport, thereby linking TCR signaling to formation of the NPC.

399
Evidence for crosstalk between NPC components and 400 Our finding that PKC-θ deficiency essentially abolished the binding of RanGAP1-SUMO1 to NPC while 419 only moderately inhibiting the sumoylation of RanGAP1 upon stimulation in vivo ( Figure 3D,E) is of interest. 420 A possible explanation for this apparent discrepancy is that the increased RanGAP1 phosphorylation itself, 421 mediated by PKC-θ upon stimulation, may also assist the binding. Moreover, other PKC-θ substrates in addition 422 to RanGAP1 may exist in the NPC complex, thus, phosphorylation of these proteins by PKC-θ may contribute 423 to the TCR-induced NPC assembly as well. The T cell adaptor protein, SLP76, has been reported to bind to 424 RanGAP1-SUMO1 and to promote the TCR-induced nuclear import of NFAT (Liu et al., 2015). Combining 425 these findings, we propose that some TCR signaling modules, e.g., PKC-θ and SLP76, translocate to the NE to 426 regulate nucleo-cytoplasmic transport and, furthermore, that PKC-θ plays an indispensable role in NPC 427

assembly. 428
The functions of PKC-θ in T cell activation and immunity are largely determined by its cellular localization 429 in the central region of the IS and by its association with CD28, two requirements for productive T cell  Interestingly, while the nuclear localization sequence of PKC-θ is known to mediate its nuclear the nucleus. It is, therefore, possible that localization of PKC-θ to the NE promotes the function of NPC, which, 445 in turn, would enable the nuclear translocation of cargo PKC-θ. We have previously demonstrated that PKC-θ 446 sumoylation, catalyzed by the SUMO E3 ligase PIASxβ, is required for its central IS localization (Wang et al., 447 2015). It remains to be determined whether PKC-θ translocation to the NE similarly requires or is dependent 448 on another mechanism. stimulation, mouse or human T cells were stimulated for various times with anti-CD3 (5 μg/ml) and/or anti-499 CD28 (2 μg/ml), which were crosslinked with goat anti-mouse IgG (10 μg/ml). 500 IP and immunoblotting. Cells washed with ice-cold PBS and lysed in lysis buffer [20 mM Tris-HCl, pH 7.5, 501 150 mM NaCl, 5 mM EDTA, 1% Nonidet-P-40, 5 mM NaPPi, 1 mM sodium orthovanadate (Na 3 VO 4 ), 1 mM 502 PMSF, and 10 μg/ml each aprotinin and leupeptin]. Whole cell lysates were incubated overnight at 4°C with the 503 indicated antibodies, and proteins were collected on protein G-Sepharose beads (GE Healthcare) for an 504 additional 4 h at 4°C with gentle shaking. The immunoprecipitated proteins were resolved by SDS-PAGE, 505 transferred onto PVDF membranes and probed with primary antibodies. Signals were visualized by enhanced 506 chemiluminescence (ECL; YESEN, Shanghai, CHINA) and films were exposed in the ChemiDoc XRS+ system 507 (Bio-Rad) or to X-ray film. Densitometry analysis was performed with ImageJ software.
ice-cold PBS were resuspended in Cytoplasmic Extraction Reagents containing proteinase inhibitors, vortexed 511 vigorously and centrifuged at 16,000 x g for 10 min. The pellet was resuspended in Nuclear Extraction Reagent 512 containing proteinase inhibitors, vortexed vigorously and centrifuged at 16,000 x g for 10 additional min. The 513 supernatant and the insoluble fraction, representing the nuclear extract and NE, respectively, were collected. 514 The NPC fraction was prepared as described (Jafferali et al., 2014), with minor modifications. Cells were 515 washed with PBS and treated with 1 mM dithiobis (succinimidyl propionate) (DSP, Sangon Biotech) in RPMI-516 1640 medium for 15 min at room temperature to crosslink the NPC. The reaction was stopped by adding 15 517 mM Tris-HCl (pH 7.4) for 10 min at room temperature. Nuclei were pelleted as described above, followed by 518 incubation in 5 volumes of 7 M urea containing 1% Triton X-100 (TX-100) and protease inhibitors for 20 min 519 on ice to resuspend the nuclei pellet. The suspension was collected as the NPC fraction, diluted 8-fold in PBS 520 containing protease inhibitors, sonicated on ice, and cleared by centrifugation at 1,000 x g for 10 min. 521 Fluorescence microscopy and analysis. Immunofluorescence was conducted as previously described (Wang et 522 al., 2015). Briefly, conjugates of Jurkat T cells and Raji APCS were plated on poly-l-lysine-coated slides, 523 incubated for 15 min at room temperature, fixed for 15 min with 4% PFA, and permeabilized with 0.2% Triton 524 X-100 for 10 min at room temperature. The slides were blocked with 2% BSA for 1 h, and samples were stained 525 with indicated antibodies overnight at 4°C. After washing with PBS, slides incubated for 1 h at room 526 temperature with secondary antibodies. After three washes with PBS, the cells were mounted with a drop of 527 mounting medium. Images were obtained with a Leica SP5 laser-scanning confocal microscope equipped with 528 Mass Spectrometric Analysis. Samples for co-IP were prepared as described previously with minor 558 modification (Wang et al., 2015). In brief, anti-CD3/CD28-stimulated Jurkat T cells were lysed, and followed 559 by IP with anti-PKC-θ or anti-IgG. IPs immobilized on protein G beads and 1 μg recombinant GST-RanGAP1 560 protein immobilized on GSH Sepharose beads were separately washed 2X with kinase buffer, and mixed to 561 initiate the kinase assay. Reactions were terminated by Laemmli sample buffer, boiled, and resolved on SDS-562 PAGE. Gel bands of interest were excised and subjected to tryptic digestion. After desalting, the peptides were 563 analyzed by tandem MS. A splitless Ultra 2D Plus system (Eksigent) coupled to the TripleTOF 5600 System 564 (AB SCIEX) with a Nanospray III source (AB SCIEX) were performed to analyze immunoprecipitated proteins 565 and identify posttranscriptional modification sites of RanGAP1. The search engine ProteinPilot V4.5 was used 566 to assigned potential modification sites with high confidence. 567 Immunoelectron microscopy. Immunoelectron microscopy was performed on Jurkat T cells stimulated for 0-568 15 min with anti-CD3 plus anti-CD28. Cells were fixed in buffer (4% paraformaldehyde, 0.2% glutaraldehyde), 569 pelleted, treated with LR white acrylic resin (L9774, Sigma-Aldrich), and then frozen for 72 h at -20°C with 570 UV irradiation. Frozen pellets were sectioned by a cryo-ultramicrotome (EM UC6 and FC6, Leica). 571 Cryosections were thawed, rinsed in PBS with 1% glycine, and incubated in 0.01 M PBS containing 0.1% BSA 572 and 5% goat serum for 30 min at room temperature. The samples were incubated with mouse anti-PKC-θ 573 antibody diluted 1:50 overnight at 4°C, and rinsed in 0.01 M PBS, then incubated with 10 nm colloidal gold-574 labeled anti-mouse IgG secondary antibody (G7652, Sigma-Aldrich) diluted 1:25 for 3 h at room temperature.
26 (Wang et al., 2015). Aliquots of T cells (3 × 10 6 ) transfected with HA-RanGAP1 or HA-RanGAP1 AA vectors 579 were stimulated for 24 h with anti-CD3 plus anti-CD28, and the concentration of IL-2 in culture supernatants 580 was determined by ELISA according to the manufacturer's instructions (BD Biosciences). 581 Computational Analysis. I-Mutant2.0 (folding.biofold.org/i-mutant/i-mutant2.0.html) was using to predict 582 RanGAP1 protein stability changes with single point mutation from the protein sequence (NP_002874.1). 583 Statistical analysis. Prism (GraphPad 5.0 Software) was used for graphs and statistical analysis. Statistical 584 analysis was performed with a two-tailed, unpaired Student's t-test or one-way ANOVA with post hoc test. P 585 values of less than 0.05 were considered statistically significant. Graphs represent mean ± standard error of 586 the mean (s.e.m). 587 588 589 590

COMPETING INTERESTS 591
The authors declare that they have no competing interests. Each symbol represents an individual T cell. Horizontal lines indicate the mean ± s.e.m. **P < 0.01 (two tailed unpaired Student's t-test).
(G) Quantification of the N/C ratio of importin β1 (left) and Ran (right) based on analysis of ~30 cells in about 6 random fields from two biological replicates of Figure 1G. Horizontal lines indicate the mean ± s.e.m. ***P < 0.001 (two tailed unpaired Student's t-test).
(H) Statistical analysis of importin β1 and Ran in nuclear extract from the experiment of Figure 1H. Analysis was based on three biological replicates. *P < 0.05 (one-way ANOVA with post hoc test). (I) Statistical analysis of importin β1 binding to NPCs in Mab414 antibody IPs from the experiment of Figure 1I. Analysis was based on three biological replicates. n.s., not significant, *P < 0.05 (oneway ANOVA with post hoc test). (J) Quantification of the N/C ratios of importin β1 (left) and Ran (right) based on analysis of ~30 cells in about 6 random fields from two biological replicates of Figure 1J. Horizontal lines indicate the mean ± s.e.m. **P < 0.01, ***P < 0.001 (two tailed unpaired Student's t-test). (K) Statistical analysis of importin β1 and Ran in nuclear extract from the experiment of Figure   1K. Analysis as based on three biological replicates. *P < 0.05 (one-way ANOVA with post hoc test). (L) Statistical analysis of importin β1 binding to NPCs (immunoprecipitated with Mab414 antibody) in the experiment shown in Figure 1L. Analysis was based on three biological replicates. n.s., not significant, *P < 0.05, **P < 0.01 (one-way ANOVA with post hoc test).   Figure 3H(E), J(F). Analysis was based on three biological replicates. n.s., not significant, **P < 0.01, ***P < 0.001 (one-way ANOVA with post hoc test). Data are representative of three biological replicates.