Abstract
During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.
Highlights
Proper progression through mitosis depends on tight regulation of protein activities in a spatial and temporal manner
We focused our efforts on SUMO2/3 as this modification is known to be more dynamic than SUMO1 during mitosis [13]
Given the specificity of RhoGDIa SUMO2/3 modification during a taxol arrest we focused on this target, as we speculated that the SUMO2/3 modification could influence changes in RhoA activity required for proper mitotic progression
Summary
Proper progression through mitosis depends on tight regulation of protein activities in a spatial and temporal manner. This regulation is mainly achieved at the level of post-translational modifications (PTMs), as this is a rapid way of changing protein activities. The very C-terminal glycine residue of SUMO is conjugated to lysine residues of target proteins. This is catalyzed by a SUMO ligase in conjunction with Ubc, which is the only E2 enzyme of the SUMO pathway [4]. A number of SUMO ligases have been described including the PIAS 1–4 proteins [5,6] and their activity is counterbalanced by a set of deSUMOylating proteins referred to as the SENPs [7]
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