SUMOylation-regulated Protein Phosphorylation, Evidence from Quantitative Phosphoproteomics Analyses

Abstract

Protein modification is critical for the regulation of protein functions. Cross-talks among different types of protein modifications should yield concerted and coordinated regulatory networks for physiological functions. Here we have employed system-wide and quantitative phosphoproteomics analyses to reveal a global cross-talk for SUMOylation-modulated phosphorylation. Furthermore, as specific examples, we have shown that the ␣ subunit of casein kinase II is SUMOylated and that this affects the phosphorylation of its substrates. SUMO-regulated phosphorylation is involved in cell cycle control. Our data demonstrate an interplay between protein SUMOylation and phosphorylation and imply a regulatory role for this SUMOylation-modulated phosphorylation.The small ubiquitin-like modifiers (SUMOs) 3 belong to a subfamily of an ubiquitin-like protein superfamily. The covalent protein modification by SUMO affects a broad range of cellular activities. There are four human SUMO genes: SUMO1, SUMO2, SUMO3, and SUMO4. SUMO2 and SUMO3 share 97% amino acid sequence identity, whereasϳ50% identity is shared by SUMO2/3 and SUMO1 (1). The SUMO2/3 proteins have the ability to form poly-SUMO chains through their internal K11 linkage, but SUMO1 does not. SUMO1 can only be conjugated to poly-SUMO2/3 chains for the termination of poly-SUMO chains (2). SUMO4 shows a similarity to SUMO2/3, but it is unclear whether it is a pseudogene.Although many advances in understanding the biochemistry of SUMOylation have been made during the past decades, progresses in large-scale identification of SUMO substrates were only made in recent years. In contrast to the traditional approach for the identification of SUMOylated proteins, which was mostly based on immunoblotting of a known protein (a hypothesis-driven process), an affinity enrichment/purification strategy coupled with mass spectrometry-based protein analysis techniques (an unbiased screening process) is currently the most widely used approach (3-5). So far, ϳ800 putative SUMO substrates have been identified (3-9). These large-scale analyses have revealed important functions of the SUMO system in many biological processes, including RNA transcription, cell cycle, mRNA processing and splicing, as well as DNA metabolism and repair. However, the exact molecular mechanism by which the SUMO system plays its role in these cellular processes is poorly understood. There is much evidence from studies of individual proteins suggesting that SUMOylation and phosphorylation processes may be connected and cross-controlled (10 -13), but whether this is a general regulatory mechanism remains to be studied.Phosphorylation is the most extensively studied reversible posttranslational protein modification. Over 77,880 non-redundant phosphorylation sites on ϳ12,000 non-redundant proteins have been identified so far, according to PhosphoSitePlus, a database compiled by Cell Signaling Technology, Inc. More than 80% of the phosphorylation sites were identified by high throughput methods, primarily the mass spectrome...

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