Novel proteomics strategy brings insight into the prevalence of SUMO-2 target sites

Abstract

Small ubiquitin-like modifier (SUMO) is covalently conjugated to its target proteins thereby altering their activity. The mammalian SUMO protein family includes four members (SUMO-1-4) of which SUMO-2 and SUMO-3 are conjugated in a stress-inducible manner. The vast majority of known SUMO substrates are recognized by the single SUMO E2-conjugating enzyme Ubc9 binding to a consensus tetrapeptide (PsiKXE where Psi stands for a large hydrophobic amino acid) or extended motifs that contain phosphorylated or negatively charged amino acids called PDSM (phosphorylation-dependent sumoylation motif) and NDSM (negatively charged amino acid-dependent sumoylation motif), respectively. We identified 382 SUMO-2 targets using a novel method based on SUMO protease treatment that improves separation of SUMO substrates on SDS-PAGE before LC-ESI-MS/MS. We also implemented a software SUMOFI (SUMO motif finder) to facilitate identification of motifs for SUMO substrates from a user-provided set of proteins and to classify the substrates according to the type of SUMO-targeting consensus site. Surprisingly more than half of the substrates lacked any known consensus site, suggesting that numerous SUMO substrates are recognized by a yet unknown consensus site-independent mechanism. Gene ontology analysis revealed that substrates in distinct functional categories display strikingly different prevalences of NDSM sites. Given that different types of motifs are bound by Ubc9 using alternative mechanisms, our data suggest that the preference of SUMO-2 targeting mechanism depends on the biological function of the substrate.

Fig. 1.

Strategy to identify heat shock-inducible SUMO-2 substrates. A, our purification is based on rapid inactivation of endogenous SUMO proteases and desumoylation of polysumoylated substrates. Endogenous SUMO proteases are inactivated immediately after cell lysis by boiling in 1% SDS. Renaturation is needed for antibody-based purification. Substrates are desumoylated on beads with recombinant Ulp-1 SUMO protease, separated by SDS-PAGE, and identified with mass spectrometry. B, K562^HA-SUMO-2 cells express His-HA-tagged SUMO-2, which is conjugated to substrate proteins upon heat shock. Lysates from untreated or heat-shocked K562^HA-SUMO-2 cells and heat-shocked parental K562 cells were separated by SDS-PAGE and detected with α-HA antibody. C, the presence of multiple sumoylation sites and the formation of SUMO-2 chains of various lengths result in substrates displaying a number of different sumoylation states. Different substrates are indicated by color codes. The different sumoylation states are split into discrete bands upon SDS-PAGE, and consequently a single band contains only a fraction of a multisumoylated substrate. Desumoylation prior to SDS-PAGE results in concentrated and well separated substrates. D, treatment with Ulp-1 SUMO protease on beads efficiently desumoylates substrates prior to SDS-PAGE. K562^HA-SUMO-2 cells were heat-shocked, SUMO-2 conjugates were purified using α-HA-agarose, and the sample was split in two. Beads were treated with or without Ulp-1, washed with PBS, and boiled in Laemmli sample buffer. Proteins were separated by SDS-PAGE and blotted with α-HA antibody. IP, immunoprecipitate.

Fig. 2.

Identification of heat shock-inducible SUMO-2 substrates. A, SUMO-2 substrates were immunoprecipitated from untreated and heat-shocked K562^HA-SUMO-2 and heat-shocked parental K562 cells. The immunoprecipitated proteins were desumoylated on beads with recombinant Ulp-1, separated by SDS-PAGE, silver-stained, and identified by LC-MS/MS. B, an aliquot from the immunoprecipitated and desumoylated samples was analyzed by Western blotting with antibodies for topoisomerase I (TopoI), DDX21, and PARP-1. C, PARP-1 undergoes SUMO-2 modification in a heat shock-inducible manner. HeLa cells transfected with Myc-tagged PARP-1 and SUMO-2 were exposed to heat shock at 43 °C for 2 h. Cells were lysed under denaturing conditions, renatured, sonicated, and centrifuged. α-Myc immunoprecipitates (IP) were separated by SDS-PAGE and blotted against SUMO-2/3.

Fig. 3.

Consensus types and GO categories in SUMO-2 substrates. A, GO categories associated with RNA-related processes are enriched in heat shock-induced SUMO-2 substrates. Heat shock-induced SUMO-2 substrates were compared with genome background for identification of positively and negatively enriched GO terms belonging to the molecular function ontology. B, many SUMO-2 substrates lack the consensus tetrapeptide. Prevalences of classical, NDSM, PDSM, and non-consensus were compared between our list of heat shock-inducible SUMO-2 substrates and genome. C, several GO categories are selectively associated with NDSM. Proportions of GO categories associated with genome, non-NDSM, classical, and NDSM were analyzed. Classical, SUMO-2 substrates containing the consensus tetrapeptide alone; NDSM, SUMO-2 substrates containing NDSM; PDSM, SUMO-2 substrates containing PDSM; non-consensus, SUMO-2 substrates lacking consensus tetrapeptide; non-NDSM, SUMO-2 substrates lacking NDSM.