A Proteomic Study of SUMO-2 Target Proteins

Abstract

The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His 6 -tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His 6 -tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach.The small ubiquitin-like modifier (SUMO) 1 is linked to target proteins by post-translational conjugation and may thereby influence protein function and/or localization (1-3). The name derives from the relationship of SUMO to the better characterized post-translational modifier ubiquitin (4). They are 18% identical at the amino acid level and show a clear similarity in their respective three-dimensional structures (5). Whereas yeast and nematodes have a single SUMO gene, in humans and mice, the SUMO family consists of three members, SUMO-1, -2, and -3, which are encoded by separate genes. The mature forms of SUMO-2 and SUMO-3 are similar (ϳ95% identical), but less closely related to SUMO-1 (ϳ50% identical) (6 -8).Despite their close similarity, there is evidence that SUMO-1 and SUMO-2/3 are preferentially conjugated to distinct sets of target proteins. Whereas RanGAP1 is modified by SUMO-1, many as yet unidentified proteins are modified by SUMO-2/3 after exposure of cells to various stress stimuli (7).Several groups independently identified SUMO-1, which is also known as PIC1, GMP1, SMT3C, UBL1, and sentrin (9 -13). In contrast to most polyubiquitinylated proteins, sumoylated proteins are not thereby targeted for degradation. SUMO-1 can even compete with ubiquitin for the same acceptor lysine in the target protein. In the case of IB␣, SUMO modification serves to protect the protein from ubiquitin-mediated degradation (14), whereas in yeast, DNA repair can proceed only after proliferating cell nuclear antigen is desumoylated to allow ubiquitination. Here, ubiquitination does not target the protein for degradation, but functions as a switch to activate the DNA repair function of proliferating cell nuclear antigen (15). Therefore, despite their structural relationship, it appears that SUMO and ubiquitin conjugation can play distinct roles in modulating target proteins. Sumoylation can also regulate the subcellular localization of target proteins. For example, SUMO conjugation is required for both the nuclear pore and kinetochore localization of RanGAP1 (1...

Regulating the regulators: Serine/arginine‐rich proteins under scrutiny

“…These results indicate that a substantial fraction of the sites seems to be unrelated to proteasomal-mediated degradation and suggests that Ub conjugation to SR proteins may work as a regulatory signal instead of as a degradation labeling (16,49). Likewise, proteomic approaches revealed that RNA-binding proteins are the predominant group among small Ubiquitin-like modifier (SUMO) conjugation substrates, including several hnRNPs, SR family members and spliceosome components (50,51). Furthermore, SUMO conjugation has been found to regulate different aspects of mRNA metabolism such as pre-mRNA 3 0 end processing and RNA editing, by modifying the function of poly(A) polymerase, symplekin and CPSF-73 in the former case and ADAR1 in the latter (52,53).…”

Sumoylation in axons triggers retrograde transport of the RNA-binding protein La

“…With the long distances that mRNAs and RNA-binding proteins must travel in neurons, sumoylation may provide a means to recycle some proteins to the nucleus by targeting these proteins for retrograde transport. Sumoylation has recently been demonstrated for a few RNAbinding proteins (19)(20)(21)(22). Sumoylation of hnRNP C decreases its nucleic acid binding (21).…”

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…Proteomic analyses have demonstrated that the pool of cellular proteins SUMOylated with SUMO1 only partially overlaps the pool of proteins SUMOylated with SUMO2/3 (47)(48)(49). Furthermore, conjugation with SUMO2/3 is known to exert at least one potentially different effect on the target from that exerted by SUMO1 conjugation, namely, the formation of SUMO2/3 chains followed by the recognition of the poly-SUMOylated target by SUMO-dependent ubiquitin ligases that polyubiquitinylate it and direct it toward proteasomal degradation, as previously reviewed (50)(51)(52).…”