The nuclear dot protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers

The Sp100 and promyelocytic leukemia proteins (PML) are constituents of nuclear domains, known as nuclear dots (NDs) or PML bodies, and are both covalently modified by the small ubiquitin-related protein SUMO-1. NDs play a role in autoimmunity, virus infections, and in the etiology of acute promyelocytic leukemia. To date, little is known about the function of the Sp100 protein. Here we analyzed Sp100 domains that determine its subcellular localization, dimerization, and SUMOylation. A functional nuclear localization signal and an ND-targeting region that coincides with an Sp100 homodimerization domain were mapped. Sequences similar to the Sp100 homodimerization/ND-targeting region occur in several other proteins and constitute a novel protein motif, termed HSR domain. The lysine residue of the Sp100 protein, to which SUMO-1 is covalently linked, was mapped within and may therefore modulate the previously described HP1 protein-binding site. A consensus sequence for SUMOylation of proteins in general is suggested. SUMOylation strictly depended on a functional nuclear localization signal but was not necessary for nuclear import or ND targeting. A three-dimensional structure of Sp100, which supports the mapping data and provides additional information on Sp100 structure/function relationships, was generated by computer modeling. Taken together, our studies indicate the existence of well defined Sp100 domains with functions in ND targeting, nuclear import, nuclear SUMOylation, and protein-protein interaction.     

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.     

Activity-dependent SUMOylation of the brain-specific scaffolding protein GISP

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.     

Enzymes of the SUMO modification pathway localize to filaments of the nuclear pore complex

SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport.     

Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.     

Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein

SUMO-1 is a member of a family of ubiquitin-like molecules that are post-translationally conjugated to various cellular proteins in a process that is mechanistically similar to ubiquitylation. To identify molecules that bind noncovalently to SUMO-1, we performed yeast two-hybrid screening with a SUMO-1 mutant that cannot be conjugated to target proteins as the bait. This screening resulted in the isolation of cDNAs encoding the b isoform of thymine DNA glycosylase (TDGb). A deletion mutant of TDGb (TDGb(Delta11)) that lacks a region shown to be required for noncovalent binding of SUMO-1 was also found not to be susceptible to SUMO-1 conjugation at an adjacent lysine residue, suggesting that such binding is required for covalent modification. In contrast, another mutant of TDGb (TDGb(KR)) in which the lysine residue targeted for SUMO-1 conjugation is replaced with arginine retained the ability to bind SUMO-1 non-covalently. TDGb was shown to interact with the promyelocytic leukemia protein (PML) in vitro as well as to colocalize with this protein to nuclear bodies in transfected cells. TDGb(KR) also colocalized with PML, whereas TDGb(Delta11) did not, indicating that the noncovalent SUMO-1 binding activity of TDGb is required for colocalization with PML. Furthermore, SUMO-1 modification of TDGb and PML enhanced the interaction between the two proteins. These results suggest that SUMO-1 functions to tether proteins to PML-containing nuclear bodies through post-translational modification and noncovalent protein-protein interaction.     

p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1

Chromosomal region maintenance 1 (CRM1) mediates p53 nuclear export. Although p53 SUMOylation promotes its nuclear export, the underlying mechanism is unclear. Here we show that tethering of a small, ubiquitin-like modifier (SUMO) moiety to p53 markedly increases its cytoplasmic localization. SUMO attachment to p53 does not affect its oligomerization, suggesting that subunit dissociation required for exposing p53’s nuclear export signal (NES) is unnecessary for p53 nuclear export. Surprisingly, SUMO-mediated p53 nuclear export depends on the SUMO-interacting motif (SIM)-binding pocket of SUMO-1. The CRM1 C-terminal domain lacking the NES-binding groove interacts with tetrameric p53, and the proper folding of the p53 core domain, rather than the presence of the N- or C-terminal tails, appears to be important for p53–CRM1 interaction. The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM. Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm. We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.     

Small ubiquitin-related modifier-1: Wrestling with protein regulation

Small ubiquitin-related modifier-1 (SUMO-1), a member of the SUMO family, is evolutionally conserved from yeast to humans. First identified in 1997, the active 97 amino acid protein conjugates to and modifies a wide variety of target proteins. Through post-translational SUMOylation of cellular proteins, SUMO-1 is involved in a myriad of biologically important events such as cell cycle progression, the maintenance of genome integrity, nuclear transport and apoptosis. Interestingly, SUMO-1 has been suggested to have the ability to act as an ubiquitin antagonist, with which it shares 18% identity. Given its wide variety of functions, it follows that alterations to this molecule could be implicated in many disease states. To date, dysregulated SUMOylation has been implicated in several neurodegenerative disorders, heart disease and cancer. This highlights not only the need for further research but also the potential of SUMO-1 as a therapeutic target.     

SUMOylation participates in induction of ischemic tolerance

Ground squirrels in hibernation torpor have been shown to have striking increases in global SUMOylation on tissue immunoblots. Here, we find evidence that global SUMOylation is also involved in ischemic tolerance in primary cortical neuronal cultures (from rats and mice) and SHSY5Y human neuroblastoma cells. Cultured cortical neurons preconditioned by sublethal oxygen/glucose deprivation (OGD) were less vulnerable to severe OGD than non-preconditioned neurons. Preconditioned neurons maintained elevated SUMO-1 conjugation levels (and, to a lesser extent those of SUMO-2/3) on western blots in contrast to non-preconditioned cells. Further, cortical neurons and SHSY5Y cells in which transfected SUMO-1 or SUMO-2 were over-expressed showed increased survival after severe OGD. In contrast, cell cultures subjected to depletion of endogenous SUMO-1 protein by RNAi had reduced survival after exposure to this form of in vitro ischemia and an attenuated protective response to preconditioning. These findings suggest that maintenance of a globally elevated SUMO-1 (and maybe SUMO-2/3) conjugation level as revealed by immunoblot assays is a component of ischemic tolerance.     

Involvement of SUMO modification in MBD1- and MCAF1-mediated heterochromatin formation

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.     

SUMOylation and PARylation cooperate to recruit and stabilize SLX4 at DNA damage sites

SUMOylation plays important roles in the DNA damage response. However, whether it is important for interstrand crosslink repair remains unknown. We report that the SLX4 nuclease scaffold protein is regulated by SUMOylation. We have identified three SUMO interaction motifs (SIMs) in SLX4, mutating all of which abrogated the binding of SLX4 to SUMO-2 and covalent SLX4 SUMOylation. An SLX4 mutant lacking functional SIMs is not recruited to PML nuclear bodies nor stabilized at laser-induced DNA damage sites. Additionally, we elucidated a novel role for PARylation in the recruitment of SLX4 to sites of DNA damage. Combined, our results uncover how SLX4 is regulated by post-translational modifications.     

The DNA topoisomerase I binding protein topors as a novel cellular target for SUMO-1 modification: characterization of domains necessary for subcellular localization and sumolation

Over the past years, modification by covalent attachment of SUMO (small ubiquitin-like modifier) has been demonstrated for of a number of cellular and viral proteins. While increasing evidence suggests a role for SUMO modification in the regulation of protein-protein interactions and/or subcellular localization, most SUMO targets are still at large. In this report we show that Topors, a Topoisomerase I and p53 interacting protein of hitherto unknown function, presents a novel cellular target for SUMO-1 modification. In a yeast two-hybrid system, Topors interacted with both SUMO-1 and the SUMO-1 conjugating enzyme UBC9. Multiple SUMO-1 modified forms of Topors could be detected after cotransfection of exogenous SUMO-1 and Topors induced the colocalization of a YFP tagged SUMO-1 protein in a speckled pattern in the nucleus. A subset of these Topors' nuclear speckles were closely associated with the PML nuclear bodies (POD, ND10). A central domain comprising Topors residues 437 to 574 was sufficient for both sumolation and localization to nuclear speckles. One SUMO-1 acceptor site at lysine residue 560 could be identified within this region. However, sumolation-deficient Topors mutants showed that sumolation obviously is not required for localization to nuclear speckles.