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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionT cells express protein-tyrosine kinases and adaptors that integrate signals for T cell activation (). Adaptors possess binding sites and discrete modular domains that integrate signals. Immune cell adaptors include SH2 domain containing leukocyte protein of 76 kDa (SLP-76) (), linker for the activation of T cells (LAT) (), and adhesion- and degranulation-promoting adaptor protein (ADAP) (). SLP-76 has a N-terminal sterile-α motif (SAM), tyrosine motifs and a SH2 domain and is needed for T cell differentiation and function (). SLP-76-deficient T cells show an impaired phospholipase Cγ1 (PLCγ1) activation and calcium mobilization (), while N-terminal residues are phosphorylated by ZAP-70 (). Y-113 and Y-128 bind exchange factor Vav1 and adaptor Nck (), resting lymphocyte kinase (Rlk) (), and inducible tyrosine kinase (Itk) (). SLP-76 binds to the SH3 domain of PLCγ1 (), while GADs SH2 domain forms a complex with LAT (). SLP-76 also forms microclusters (), exerts feedback control on ZAP-70 (), and interacts with subsynaptic LAT clusters (). The SLP-76 SH2 domain binds to ADAP (href="#bib11" rid="bib11 bib31" class=" bibr popnode">da Silva et al., 1997; Musci et al., 1997) and hematopoietic progenitor kinase-1 (HPK-1) (href="#bib12" rid="bib12 bib49" class=" bibr popnode">Di Bartolo et al., 2007; Shui et al., 2007). In turn, ADAP binds to adaptor SKAP1 (SKAP-55) for integrin adhesion (href="#bib39" rid="bib39 bib40 bib53" class=" bibr popnode">Raab et al., 2010, 2011; Wang and Rudd, 2008).SLP-76 is also needed downstream to activate transcription factors NFAT (nuclear factor for the activation of T cells) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) (href="#bib58" rid="bib58" class=" bibr popnode">Yablonski et al., 1998). NFAT possesses two basic nuclear localization sequences (NLSs) for nuclear import dependent on dephosphorylation by calcineurin (href="#bib30" rid="bib30 bib57" class=" bibr popnode">Müller and Rao, 2010; Wu et al., 2007). Dephosphorylation unmasks nuclear-location signals (href="#bib48" rid="bib48" class=" bibr popnode">Shibasaki et al., 1996). Similarly, NF-κB plays roles in inflammation, cell activation, and differentiation (href="#bib13" rid="bib13 bib47" class=" bibr popnode">Ghosh and Karin, 2002; Sen, 2011). Coreceptor CD28 and innate receptors activate NF-κB transcription via different pathways in T cells (href="#bib27" rid="bib27 bib51" class=" bibr popnode">Marinari et al., 2002; Thaker et al., 2015).Nuclear transport is mediated by the nuclear pore complex (NPC) (href="#bib8" rid="bib8 bib16" class=" bibr popnode">Chatel and Fahrenkrog, 2012; Hoelz et al., 2011). The NPC is composed of more than 30 nucleoporins (Nups) needed for anchorage and the formation of a central mesh in the channel (href="#bib1" rid="bib1 bib10" class=" bibr popnode">Allen et al., 2008; D’Angelo and Hetzer, 2008). Intriguingly, eight filaments extend into the cytoplasm comprised of RanBP2 (Nup358) and RanGAP1, the latter having GTPase activity for GTP-Ran (href="#bib3" rid="bib3" class=" bibr popnode">Bischoff et al., 1994). This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier) (href="#bib21" rid="bib21 bib24" class=" bibr popnode">Lee et al., 1998; Mahajan et al., 1997). Ran binding to GTP causes importins to release protein in the nucleus, while nonhydrolysable GTP accumulates Ran-GTP at the filaments (href="#bib29" rid="bib29" class=" bibr popnode">Melchior et al., 1995). RanBP2/RanGAP1 and associated SUMO1/Ubc9 form a multisubunit SUMO E3 ligase (href="#bib33" rid="bib33 bib54" class=" bibr popnode">Pichler et al., 2002; Werner et al., 2012).SLP-76 microclusters at the cell surface translocate to the perinuclear region of T cells (href="#bib7" rid="bib7" class=" bibr popnode">Bunnell et al., 2002). While adaptors mediate TCR proximal signaling, direct regulation of the NPC has not been reported. Here, we show that direct SLP-76 binding to the SUMO-RanGAP1 of cytoplasmic filaments of the NPC is required for the regulation of transcription factor entry into the nucleus of T cells. Our findings identify a surprising direct mechanism of NPC regulation by an immune adaptor in T cells.