Structural and dynamic independence of isopeptide-linked RanGAP1 and SUMO-1

Although sumoylation regulates a diverse and growing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established. To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1. Based on amide chemical shift and 15N relaxation measurements, we show that the C terminus of SUMO-1 and the loop containing the consensus sumoylation site in RanGAP1 are both conformationally flexible. Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys524 in RanGAP1 to the C-terminal Gly97 of SUMO-1. Therefore, SUMO-1 and RanGAP1 behave as "beads-on-a-string," connected by a flexible isopeptide tether. Accordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage. We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries.     

In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.     

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1–6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif. (Mol Cell Biochem 271: 215–223, 2005)     

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.     

Pellino-1, an adaptor protein of interleukin-1 receptor/toll-like receptor signaling,is sumoylated by Ubc9

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.     

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.     

类泛素家族SUMO-1和UBC9的克隆、融合表达及纯化

目的：克隆类泛素化家族SUMO-1和UBC9基因,表达并纯化二者与谷胱甘肽S-转移酶（GST）的融合蛋白。方法：用PCR方法从乳腺文库中扩增SUMO-1和UBC9的编码序列,分别将其以正确相位与pGEX-KG载体中的GST编码序列融合,得到重组质粒pGST-SUMO-1和pGST-UBC9,分别转化大肠杆菌DH5α,表达融合蛋白GST-SUMO-1和GST-UBC9;用谷胱甘肽-Sepharose4B亲和纯化融合蛋白;用Western印迹检测融合蛋白的表达及纯化。结果：分别构建了SUMO-1和UBC9的融合表达载体;Western印迹检测表明,GST-SUMO-1和GST-UBC9融合蛋白获得表达;纯化得到了融合蛋白。结论：克隆、表达并纯化了SUMO-1和UBC9与GST的融合蛋白。