Annette Flotho

Sumoylation: A Regulatory Protein Modification in Health and Disease

Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis.

Ubc9 Sumoylation Regulates Sumo Target Discrimination.

Posttranslational modification with small ubiquitin-related modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single E1, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9 approximately SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases.

The Nup358-RanGAP Complex Is Required for Efficient Importin α/β-dependent Nuclear Import

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.

The RanBP2/RanGAP1∗SUMO1/Ubc9 Complex Is a Multisubunit SUMO E3 Ligase

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.

Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies

SUMOylation is a protein modification that regulates the function of hundreds of proteins. Detecting endogenous SUMOylation is challenging: most small ubiquitin-related modifier (SUMO) targets are low in abundance, and only a fraction of a proteins cellular pool is typically SUMOylated. Here we present a step-by-step protocol for the enrichment of endogenous SUMO targets from mammalian cells and tissues (specifically, mouse liver), based on the use of monoclonal antibodies that are available to the scientific community. The protocol comprises (i) production of antibodies and affinity matrix, (ii) denaturing cell lysis, and (iii) SUMO immunoprecipitation followed by peptide elution. Production of affinity matrix and cell lysis requires ∼1 d. The immunoprecipitation with peptide elution can be performed in 2 d. As SUMO proteins are conserved, this protocol should also be applicable to other organisms, including many vertebrates and Drosophila melanogaster.

The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2s SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2s Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions.

Recombinant Reconstitution of Sumoylation Reactions In Vitro

Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.

The RanBP2/RanGAP1*SUMO1/Ubc9 complex: A multisubunit E3 ligase at the intersection of sumoylation and the RanGTPase cycle

Posttranslational modification of proteins with SUMO and the RanGTP/GDP cycle are two essential cellular mechanisms contributing directly or indirectly to almost every cellular event. The SUMO E3 ligase RanBP2 (Nup358) and the Ran GTPase activating protein (RanGAP1) are known to form a stable complex throughout the cell cycle suggesting a link between sumoylation of proteins and RanGTP hydrolysis. In a recent study we demonstrated that the stable complex of RanBP2, sumoylated RanGAP1 and Ubc9 (and not RanBP2 by itself) represents the physiologically relevant form of the SUMO ligase. Characterization of the interactions reveals an intricate proximity of two catalytic activities, sumoylation and RanGTP hydrolysis. In this ExtraView we summarize our results and discuss some ideas about a potential coupling of both processes.

The Ran GTPase-activating protein (RanGAP1) is critically involved in smooth muscle cell differentiation, proliferation and migration following vascular injury: implications for neointima formation and restenosis.

Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50% induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction of RanGAP1 levels also resulted in a reduction of mitogen induced cellular migration and proliferation as well as a significant upregulation of the cyclin-dependent kinase inhibitor p27KIP1, without evidence for cellular necrosis. These findings suggest that RanGAP1 plays a critical role in smooth muscle cell differentiation, migration and proliferation in vitro and in vivo. Appropriate modulation of RanGAP1 expression may thus be a strategy to modulate VPD development such as restenosis.