The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

Abstract

SummaryWhile immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

Ubc9 Binds to ADAP and Is Required for Rap1 Membrane Recruitment, Rac1 Activation, and Integrin-Mediated T Cell Adhesion

“…Jurkat T cells and murine T cell hybridoma DC27.10 and 2B4 cells were maintained in RPMI 1640 medium supplemented with 5% (v/v) FBS (Sigma) and 100 U/ml penicillin/streptomycin (29). Total CD4 + T cells were enriched from primary T cells from 6 to 8-wk-old female C57BL/6J mice (Shanghai Laboratory Animal Center, Shanghai, China) with mouse CD4 + enrichment selection column (R&D System) according to the manufacturer's manual.…”

“…Cells were incubated with fluorescence-labeled Abs (BD Biosciences) for 1 h at room temperature in FACS buffer (0.5% BSA in PBS), washed three times with PBS, and resuspended into FACS buffer. Analyses were performed using a FACSCalibur flow cytometer (BD Biosciences) as described before (29). For the intracellular Ca 2+ measurement, cells were loaded with 5 mM of Indo-1-AM (Thermo Fisher) in RPMI 1640 and incubated for 1 h at 37˚C.…”

“…Immunofluorescence microscopy was conducted as described previously (29). Cells were seeded on poly-L-lysine (Sigma) -coated slides, fixed in 4% paraformaldehyde for 15 min, permeabilized with 90% methanol for 30 min, and blocked with 5% BSA for 1 h. Cells were subsequently stained with the indicated primary Abs in blocking buffer overnight and visualized with Alexa Fluor 488-conjugated goat anti-mouse or Alexa Fluor 555conjugated donkey anti-rabbit Abs (Cell Signaling Technology) correspondingly.…”

Itk promotes the integration of TCR and CD28 costimulation, through its direct substrates, SLP-76 and Gads

“…Gads may directly influence transcriptional events via the ability of SLP-76 to translocate to the nuclear pores, where it regulates the nuclear translocation NFκB [86]. It remains to be seen whether this activity depends on Itk-targeted phosphorylation sites on the adaptors.…”

Mice Deficient in Nucleoporin Nup210 Develop Peripheral T Cell Alterations

“…Further studies have suggested that Nup210 is acting as a scaffolding protein for transcriptional complexes such as Mef2C, and that the tissue-specific expression is most likely a driver for the specialization of NPCs in different cell types, thereby playing a role in the regulation of cell fate ( 17 ). The regulatory role of the NPC in the import of transcription factors in T cells in the context of inflammation and immunity has been shown in a number of studies ( 18 – 21 ), and this cellular function has also been studied in detail in myocyte culture ( 17 ), however the role of NUP210 at the organism level has not previously been studied.…”