SUMOs (small ubiquitin-related modifiers), are ~100 amino acid proteins that are post-translationally and covalently conjugated to other proteins (Johnson, 2004
; Kerscher et al., 2006
). Although invertebrates express one SUMO, vertebrates express three paralogues: SUMO-1, SUMO-2 and SUMO-3. Human SUMO-2 and SUMO-3 are ~96% identical to each other (and are referred to collectively as SUMO-2/3), whereas they share only ~45% identity with SUMO-1. All three SUMOs are covalently conjugated to other proteins through a common enzyme cascade involving the same E1 activating and E2 conjugating enzymes (Johnson, 2004
). In addition, all three paralogues are generally thought to affect modified proteins through related mechanisms involving effects on protein structure and function and/or changes in protein-protein or nucleic acid interactions. Several lines of evidence, however, indicate that SUMO-2/3 have protein targets, signaling properties and functions that are unique from those of SUMO-1. Proteomic studies, for example, have identified distinct but partially overlapping subsets of SUMO-1 and SUMO-2/3 modified proteins (Rosas-Acosta et al., 2005
; Vertegaal et al., 2006
). In addition, SUMO-2/3 conjugation is preferentially up-regulated in response to cell stress and SUMO-2/3 are more mobile within the nucleus relative to SUMO-1 (Ayaydin and Dasso, 2004
; Saitoh and Hinchey, 2000
). Despite these general observations, however, specific functional differences between SUMO-1 and SUMO-2/3 remain to be identified.
Over 200 proteins have been identified as SUMO-1 or SUMO-2/3 substrates through biochemical and proteomic approaches, implicating SUMOylation as a regulator of a wide range of functions largely associated with the nucleus (Seeler and Dejean, 2003
). Genetic studies in particular have identified roles for SUMOylation in regulating chromosome segregation and progression through mitosis. In yeast and Drosophila, SUMOylation is essential for mitotic chromosome condensation, sister chromatid cohesion, kinetochore function and mitotic spindle elongation (Watts, 2007
). Although it can be presumed that multiple different proteins are SUMOylated at distinct stages to regulate these diverse mitotic events, few relevant SUMO substrates have been identified. Known substrates include topoisomerase II, Pds5, Ndc10 and Bir1 (Azuma et al., 2003
; Bachant et al., 2002
; Montpetit et al., 2006
; Stead et al., 2003
). Notable, however, is that few studies have identified mitotic functions or specific protein targets for SUMOylation in mammalian cells.
Here, we demonstrate that SUMO-1 and SUMO-2/3 conjugation to distinct subsets of proteins is essential for progression through mitosis in mammalian cells. Inhibition of SUMOylation caused cells to arrest at prometaphase due to a defect in the association of CENP-E, a SUMO-2/3 substrate and SUMO-2/3 polymeric chain binding protein, to kinetochores. Our findings reveal a paralogue-specific role for SUMO-2/3 in regulating the association of CENP-E with kinetochores and demonstrate that SUMOylation, like ubiquitination and phosphorylation, is a key regulator of mitosis.