Dataset Information

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis

ABSTRACT: Successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes. Biochemical analysis of cytokinesis is challenging due to its dynamic and rapid nature that necessitates the establishment of robust methodologies. Our comparative proteomic approach suggests that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-suited system for global biochemical analysis in mammalian cells. We established a phosphoproteomic signature of cytokinesis. More than 10,000 phosphorylation sites were systematically screened and monitored; around 800 of these phosphosites were up-regulated during cytokinesis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Erdem Sanal

LAB HEAD: Nurhan Özlü

PROVIDER: PXD008451 | Pride | 2018-02-07

REPOSITORIES: Pride

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Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis.

Karayel Özge Ö Şanal Erdem E Giese Sven H SH Üretmen Kagıalı Zeynep Cansu ZC Polat Ayşe Nur AN Hu Chi-Kuo CK Renard Bernhard Y BY Tuncbag Nurcan N Özlü Nurhan N

Scientific reports 20180202 1

The successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes that together form partly redundant pathways, depending on the cell type. The biochemical analysis of this process is challenging due to its dynamic and rapid nature. Here, we systematically compared monopolar and bipolar cytokinesis and demonstrated that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-su ...[more]

PMID: 29396449

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Project description:SUMO modification of proteins (sumoylation) is essential for mitotic progression from yeast to humans, but only a limited number of sumoylated proteins with functions in mitosis have been discovered. Vertebrates express three SUMO paralogs, SUMO-1, SUMO-2 and SUMO-3, but only SUMO-2 and -3 are chromosome-associated in mitosis. In this study, we used chromosome spreads to more precisely define the localization of endogenous SUMO-2 and -3 to the centromere as well as the chromosome protein scaffold. Furthermore, we developed methodologies for the immunopurification of endogenous SUMO-2 and -3 modified proteins from cell extracts. Using LC-MS/MS, we analyzed proteins immunopurified from mitotic chromosome fractions and G0 nuclear fractions. We identified 296 putative sumoylated proteins, with 138 being modified specifically in mitosis. We identified proteins known to localize to the centromere and kinetochore and the chromosome protein scaffold, consistent with SUMO-2/3 localization. Furthermore, we demonstrate that utilizing cell synchronization and fractionation allowed for the discovery of low abundance sumoylated proteins that are not detected in large asynchronous analyses. Our results provide a foundation for further characterization of the roles of sumoylation in regulating diverse aspects of chromosome segregation in mitosis. Thermo .raw files were uploaded to the ProHits (Liu et al, 2010) analytical suite and converted to .mzXML format using ReAdW software. Data were searched using X!Tandem (Craig & Beavis, 2004) against human ORFs (RefSeq v45). Search parameters specified a parent MS tolerance of +/- 15ppm, and an MS/MS tolerance of 0.4 Da, with up to two missed cleavages for trypsin. Oxidation of methionine and tryptophan, ubiquitylation of lysine, and alkylation of cysteine (by NEM) were allowed as variable modifications. Statistical validation of the results was performed using Peptide Prophet and Protein Prophet (Keller et al, 2002; Nesvizhskii et al, 2003) as part of the trans-proteomic pipeline. For each search, the Protein Prophet probability at a 1% false discovery rate was used as a cutoff value to generate SAINT-compatible input files. SAINT parameters were as follows: 5000 iterations, low mode Off (0), minFold 1 and normalization (1) (Choi et al, 2011). SAINT cutoff values of 0.75 were used to generate a high-confidence list of protein identifications.

Dataset Information

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis

Publications

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis.

Similar Datasets

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