Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 Ligase Complex

The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2''s E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP''s affinity to RanBP2''s RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2''s RBDs, which is prevented by the transport factor NTF2.     

Thyroid Hormone Receptor Isoform-specific Modification by Small Ubiquitin-like Modifier (SUMO) Modulates Thyroid Hormone-dependent Gene Regulation

Thyroid hormone receptor (TR) α and β mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRβ plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRβ at lysines 50, 146, and 443. Sumoylation of TRβ was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxβ and TRβ-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHβ-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHβ-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.