Mitotic Substrates of the Kinase Aurora with Roles in Chromatin Regulation Identified Through Quantitative Phosphoproteomics of Fission Yeast

Abstract

Kinases of the Aurora family are essential for the proper execution of mitosis in eukaryotes, and Aurora inhibitors are in clinical trials as anticancer drugs. We applied site-specific quantitative phosphoproteomics in conjunction with chemical inhibition of Aurora to identify mitotic Aurora substrates in fission yeast on a proteome-wide scale. We detected 8000 phosphorylation events, of which we assigned almost 6000 to a specific residue; 220 were reduced in cells exposed to the Aurora inhibitor. After controlling for unspecific effects of the inhibitor, we classified 70 sites (on 42 proteins) as probable targets of Aurora, which enabled refinement of the consensus sequence for phosphorylation by Aurora. Several of the substrate candidates were known targets of Aurora, validating the approach, but most represented newly detected Aurora substrates. The involvement of these Aurora substrates in diverse aspects of chromatin dynamics suggests that in addition to its established role in controlling chromosome compaction and attachment to the mitotic spindle, Aurora influences other aspects of chromatin architecture and function during mitosis.

Role and regulation of kinesin-8 motors through the cell cycle

“…The binding sites for type-1-phosphatase (PP1) and EB1 are shown in orange and black, respectively. Green lines indicate in vivo protein phosphorylation sites identified by mass spectrometry (www.phosphosite.org; www.phos phogrid.org; Koch et al 2011). (Color figure online)…”

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast

“…Because wild-type Schizosaccharomyces pombe can synthesize all amino acids, labeling cells with heavy amino acids requires the use of auxotrophic strains. Labeling fission yeast lysine-auxotrophic cells with heavy lysine is straightforward and does not present any problems (Koch et al 2011;Wang et al 2012). However, arginine is catabolized and converted into other amino acids, including but not limited to proline.…”

“…In fission yeast, arginine conversion is much more extensive (Bicho et al 2010) and thus must be dealt with more decisively. Two general approaches are possible, and both have been used successfully (Bicho et al 2010;Koch et al 2011). The first approach (Koch et al 2011) avoids heavy arginine altogether.…”

“…Two general approaches are possible, and both have been used successfully (Bicho et al 2010;Koch et al 2011). The first approach (Koch et al 2011) avoids heavy arginine altogether. Instead, cells are labeled only with heavy lysine, and, in place of trypsin, proteolytic digestion into peptides is performed using lysyl endopeptidase Lys-C, which cleaves after lysine residues.…”

“…We describe construction of fission yeast strains suitable for SILAC MS, as well as conditions for robust growth, harvesting, and processing of cells (Bicho et al 2010;Koch et al 2011) ). This strategy has been used in several large-scale phosphoproteomics studies in eukaryotes (Olsen et al 2006 and prokaryotes (Maček et al 2007(Maček et al , 2008.…”