Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes

Abstract

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 to the inner-centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere and kinetochore associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs.

A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis

“…This localization pattern is reminiscent of that of SUMO-1 and SUMO-2/3 during mitosis (Zhang et al., 2008), and the only SUMO isoform in nematodes displays a combination of these localization patterns during the first embryonic mitotic division (Pelisch et al., 2014). Many SUMO substrates are involved in chromatin structure and function (Cubeñas-Potts and Matunis, 2013), and KIF4A, the human ortholog of KLP-19, has been identified as a SUMO substrate in mitotic chromatin (Cubeñas-Potts et al., 2015). These observations support the notion that while precise mechanisms that guarantee proper chromosome orientation, congression, and segregation might differ between meiosis and mitosis and also among species, SUMO is likely a key contributor to a timely and accurate regulation of protein interactions within narrow spatial and temporal windows.…”

SUMO conjugation – a mechanistic view

“…In recent years, several efforts have been made to study sumoylation on a global and site-specific manner by high resolution mass spectrometry (14,15,(51)(52)(53)(54). Reanalyzing all these available data sets (13) revealed that at physiological conditions, at least half of the SUMO substrates are modified at the minimal KxE motif, although upon stress, more lysines at non-SCM sites are modified.…”

“…Indeed, more than 1000 sumoylated proteins have been identified, with the numbers continuously increasing (13). Sumoylation is highly dynamic and the global SUMO proteome is constantly changing, for example, during cell cycle progression and cell differentiation, and it is drastically induced upon stress (14)(15)(16)(17). Such stress stimuli include DNA damage, heat shock, proteasomal inhibition, viral infection or ischemic challenge.…”

SUMOylation-Mediated Regulation of Cell Cycle Progression and Cancer

“…These proteomic approaches have also greatly helped in identifying other SUMO target proteins essential for cell cycle progression and have uncovered global SUMO dynamics throughout the cell cycle [23,55]. It would be interesting to identify groups of specific substrates regulated by the different dynamic SUMO machinery components.…”

“…Additional CDKs and cyclins have been identified as SUMO targets in recent proteomic screens and targeted approaches, including CDK1, CDK9, CDK11 and Cyclin-E, further highlighting crosstalk between SUMOylation and phosphorylation during cell cycle regulation [23,48,54] (Figure 2). These proteomic approaches have also greatly helped in identifying other SUMO target proteins essential for cell cycle progression and have uncovered global SUMO dynamics throughout the cell cycle [23,55]. It would be interesting to identify groups of specific substrates regulated by the different dynamic SUMO machinery components.…”