Broad Spectrum Identification of Cellular Small Ubiquitin-related Modifier (SUMO) Substrate Proteins

Abstract

Reversible covalent modification of proteins with a small ubiquitin-related modifier (SUMO) is emerging as an important system contributing to dynamic regulation of protein function. To enhance our understanding of the cell regulatory systems impacted by sumoylation, we used affinity chromatography-coupled high pressure liquid chromatography/tandem mass spectrometry for unbiased identification of candidate cellular SUMO substrate proteins. Here we describe the identification of 21 candidate sumoylated proteins from whole-cell lysates of HEK-293 cells. The nature of the proteins identified is consistent with a role for sumoylation in diverse cell regulatory systems but highlights regulation of chromatin organization and gene expression as major systems targeted by the sumoylation machinery.In addition to the well recognized role of protein phosphorylation, dynamic post-translational regulation of protein function can be achieved through a combination of a variety of other reversible covalent modifications including methylation, acetylation, ubiquitination, and sumoylation. Ubiquitination and sumoylation involve the ligation of ubiquitin family small polypeptides to one or more lysines of the target protein. Ubiquitination has long been recognized as an address code that sends proteins to the proteasome for degradation. However, apart from its role in protein degradation, ubiquitination also participates in the regulation of gene expression, signal transduction, and intracellular transport. The consequences of sumoylation on target proteins are relatively obscure; although based on the growing list of identified sumoylated proteins, sumoylation has been implicated in a diverse array of cell regulatory functions including the regulation of chromatin structure, subcellular compartmentalization, transcription factor activity, DNA binding, and protein complex assembly (1-3).There are three known small ubiquitin-like modifier (SUMO) 1 family members in mammalian cells: SUMO1, -2, and -3. The biochemical pathway mediating protein sumoylation has been characterized and is analogous to that defined for ubiquitination. Namely, the combined action of three proteins (an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase) mediates isopeptide bond formation between SUMO and the target lysine on the substrate protein. Importantly, sumoylation is reversible, and a number of SUMO proteases that can cleave SUMO from specific target proteins have been identified (1-3).The bulk of present knowledge of the role of sumoylation in the regulation of cell behavior has been inferred from the nature of identified SUMO substrate proteins (3). The list of known SUMO substrates has been compiled by a combination of serendipitous observations and direct candidate analysis. It is not at all clear how well this list reflects the contribution of dynamic sumoylation to cell regulatory events. Therefore, we wished to assess the feasibility of a proteomics-based approach for an unbiased sampling of cellular SUMO target proteins. Here, ...

A Proteomic Study of SUMO-2 Target Proteins

“…In line with our results, SUMO-2 was recently identified in a purified SUMO-1 fraction (72). In this study, hemagglutinin-SUMO-1 was used in an unbiased approach to identify SUMO-1 targets.…”

A Proteome-wide Approach Identifies Sumoylated Substrate Proteins in Yeast

“…Nevertheless, both the methods could be useful to analyze SUMO modification in protein complexes and follow the changes of sumoylation patterns during formation and turnover of these large multimeric assemblies. Interestingly, the human homologues of the yeast RNA polymerase subunits Rpc160, Rpc128, Rpc82, Rpc37, Rpc25, Rpac40, and Rpabc27 are found to be "potentially" sumoylated in a proteomic approach using mammalian cells (10). Thus, sumoylation of RNA polymerases is conserved, suggesting that this post-translational modification is important for gene expression.…”

“…Although we could not unravel the entire SUMO proteome, the potent affinity purification of Smt3-ProtA in combination with mass spectrometry was able to identify several unknown SUMO substrates. Indeed similar proteomic approaches in mammalian cells lead to the isolation of a distinct set of proteins (10,12). It is thus conceivable that only several different proteomic approaches will give a more comprehensive list of sumoylated proteins.…”

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

“…The level of H3K9 at the SUMO-2-repressed promoter has not been determined, but increased methylation may be anticipated, because HP1, an H3 methyl K9 binding protein, is recruited to a Gal4-dependent promoter by Gal4-Ubc9 (29) Of the SUMO-2-interacting proteins assayed (RanGAP1, LSD1, SETDB1, PELP1, and NXP-2), only NXP-2 and LSD1 repressed when tethered to a promoter as fusions to the Gal4 DNA-binding domain. NXP-2, was identified in an immunoscreen of a cDNA library for nuclear matrix proteins, interacts with SUMO-1 by yeast two-hybrid (30), and is SUMO modified (31).…”