Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration.

Proteomics studies have revealed that SUMOylation is a widely used post-translational modification (PTM) in eukaryotes. However, how SUMO E1/2/3 complexes use different SUMO isoforms and recognize substrates remains largely unknown. Using a human proteome microarray-based activity screen, we identified over 2500 proteins that undergo SUMO E3-dependent SUMOylation. We next constructed a SUMO isoform- and E3 ligase-dependent enzyme-substrate relationship network. Protein kinases were significantly enriched among SUMOylation substrates, suggesting crosstalk between phosphorylation and SUMOylation. Cell-based analyses of tyrosine kinase, PYK2, revealed that SUMOylation at four lysine residues promoted PYK2 autophosphorylation at tyrosine 402, which in turn enhanced its interaction with SRC and full activation of the SRC-PYK2 complex. SUMOylation on WT but not the 4KR mutant of PYK2 further elevated phosphorylation of the downstream components in the focal adhesion pathway, such as paxillin and Erk1/2, leading to significantly enhanced cell migration during wound healing. These studies illustrate how our SUMO E3 ligase-substrate network can be used to explore crosstalk between SUMOylation and other PTMs in many biological processes.

Global Analysis of SUMO-Binding Proteins Identifies SUMOylation as a Key Regulator of the INO80 Chromatin Remodeling Complex.

SUMOylation is a critical regulator of a broad range of cellular processes, and is thought to do so in part by modulation of protein interaction. To comprehensively identify human proteins whose interaction is modulated by SUMOylation, we developed an in vitro binding assay using human proteome microarrays to identify targets of SUMO1 and SUMO2. We then integrated these results with protein SUMOylation and protein-protein interaction data to perform network motif analysis. We focused on a single network motif we termed a SUMOmodPPI (SUMO-modulated Protein-Protein Interaction) that included the INO80 chromatin remodeling complex subunits TFPT and INO80E. We validated the SUMO-binding activity of INO80E, and showed that TFPT is a SUMO substrate both in vitro and in vivo We then demonstrated a key role for SUMOylation in mediating the interaction between these two proteins, both in vitro and in vivo By demonstrating a key role for SUMOylation in regulating the INO80 chromatin remodeling complex, this work illustrates the power of bioinformatics analysis of large data sets in predicting novel biological phenomena.

E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex.

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (kobs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of kobs on TDG concentration gives kmax = 1.6 min(-1) and K1/2 = 0.55 µM, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (Kd ∼50 nM). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.

SUMO binding by the Epstein-Barr virus protein kinase BGLF4 is crucial for BGLF4 function.

An Epstein-Barr virus (EBV) protein microarray was used to screen for proteins binding noncovalently to the small ubiquitin-like modifier SUMO2. Among the 11 SUMO binding proteins identified was the conserved protein kinase BGLF4. The mutation of potential SUMO interaction motifs (SIMs) in BGLF4 identified N- and C-terminal SIMs. The mutation of both SIMs changed the intracellular localization of BGLF4 from nuclear to cytoplasmic, while BGLF4 mutated in the N-terminal SIM remained predominantly nuclear. The mutation of the C-terminal SIM yielded an intermediate phenotype with nuclear and cytoplasmic staining. The transfer of BGLF4 amino acids 342 to 359 to a nuclear green fluorescent protein (GFP)-tagged reporter protein led to the relocalization of the reporter to the cytoplasm. Thus, the C-terminal SIM lies adjacent to a nuclear export signal, and coordinated SUMO binding by the N- and C-terminal SIMs blocks export and allows the nuclear accumulation of BGLF4. The mutation of either SIM prevented SUMO binding in vitro. The ability of BGLF4 to abolish the SUMOylation of the EBV lytic cycle transactivator ZTA was dependent on both BGLF4 SUMO binding and BGLF4 kinase activity. The global profile of SUMOylated cell proteins was also suppressed by BGLF4 but not by the SIM or kinase-dead BGLF4 mutant. The effective BGLF4-mediated dispersion of promyelocytic leukemia (PML) bodies was dependent on SUMO binding. The SUMO binding function of BGLF4 was also required to induce the cellular DNA damage response and to enhance the production of extracellular virus during EBV lytic replication. Thus, SUMO binding by BGLF4 modulates BGLF4 function and affects the efficiency of lytic EBV replication.

Lysyl-tRNA synthetase interacts with EF1alpha, aspartyl-tRNA synthetase and p38 in vitro.

The functions of evolved mammalian supramolecular assemblies and extensions of enzymes are not well understood. Human lysyl-tRNA synthetase (hKRS) only upon the removal of the amino-terminal extension (hKRSDelta60) bound to EF1alpha and was stimulated by EF1alphain vitro. HKRS and hKRSDelta60 were also differentially stimulated by aspartyl-tRNA synthetase (AspRS) from the multi-synthetase complex. The non-synthetase protein from the multi-synthetase complex p38 alone did not affect hKRS lysylation but inhibited the AspRS-mediated stimulation of hKRS. These results revealed the functional interactions of hKRS and shed new lights on the functional significance of the structural evolution of multienzyme complexes and appended extensions.

Identification of SUMO E3 ligase-specific substrates using the HuProt human proteome microarray.

The functional protein microarray is a powerful and versatile systems biology and proteomics tool that allows the rapid activity profiling of thousands of proteins in parallel. We have recently developed a human proteome array, the HuProt array, which includes ~80 % of all the full-length proteins of the human proteome. In one recent application of the HuProt array, we identified numerous SUMO E3 ligase-dependent SUMOylation substrates. For many SUMO E3 ligases, only a small number of substrates have been identified and the target specificities of these ligases therefore remain poorly defined. In this protocol, we outline a method we developed using the HuProt array to screen the human proteome to identify novel SUMO E3 ligase substrates recognized by specific E3 ligases.

Characterization of the SUMO-binding activity of the myeloproliferative and mental retardation (MYM)-type zinc fingers in ZNF261 and ZNF198.

SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.

E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1.

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

SUMO, PTEN and Tumor Suppression.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified in 1997 as a tumor suppressor gene through mapping of homozygous mutations occurring in multiple sporadic tumor types and in patients with cancer predisposition syndromes including Cowden disease (Song et al., 2012). Since that time, PTEN has emerged as one of the most frequently mutated or deleted genes in human cancers, including human skin cancers. In particular, loss of PTEN function through mutation or deletion has been observed in up to 70% of melanoma cell lines, and epigenetic silencing of PTEN has been observed in 30-40% of malignant melanomas (Mehnert and Kluger, 2012).

RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage.

The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification.

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are approximately 96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralog-selective modification.

Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli.

Fusion and affinity tags are popular tools for the expression of mammalian proteins in bacteria. To facilitate the selection of expression approaches, a systematic comparison was performed. We cloned, sequenced, and expressed in Escherichia coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. The tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Similar enhancement of hDRS activity was obtained by fusion to ubiquitin or SUMO. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity. Fusion proteins were specifically cleaved without altering the N-terminal of hDRS. After cleavage hDRS remained soluble and active with a specific activity comparable to that of the fused protein. Similar activity was observed with biotin- and 6xHis-tagging of hDRS. Higher purity but significantly lower yields of hDRS were obtained using biotin-tagging. Overall we demonstrated ubiquitin and SUMO fusion proteins similarly enhanced the proper folding of hDRS expressed in E. coli. In comparison to previous expressions of hDRS as a GST fusion, ubiquitin, and SUMO fusions provided higher yields and easier purification and cleavage.