A quantitative atlas of mitotic phosphorylation

Abstract

The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G 1 and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.cell cycle ͉ cyclin-dependent kinase ͉ mass spectrometry ͉ proteomics

Rictor is a novel target of p70 S6 kinase-1

“…In some cases, definitive localization of the phosphorylation site could not be obtained (Figure 1a, sites marked in green). Some of these sites have previously been reported, including Ser21 and Ser1177, which have been identified in different largescale analyses of phosphoproteins (Beausoleil et al, 2004;Dai et al, 2007;Cantin et al, 2008;Daub et al, 2008;Dephoure et al, 2008;Zanivan et al, 2008). In addition, the phosphopeptides containing Ser1174, Ser1219, Thr1222, Thr1295, Ser1385, Tyr1386 and Ser1388, which have recently reported been, were also detected (Dephoure et al, 2008;Zanivan et al, 2008).…”

Mitotic phosphorylation of CCCTC‐binding factor (CTCF) reduces its DNA binding activity

“…Supernatant and pellet fractions were obtained by centrifugation at 2300 g for 5 min. [9] and [10]. Blue and green characters indicate phosphorylation sites analyzed in [11] and [17], respectively.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Some of these sites may be more suitable markers of catalytic activation. identify proteins whose phosphorylation state is regulated by the cell cycle [102][103][104] and (c) indentify proteins whose phosphorylation state is perturbed as a consequence of drug/inhibitor treatment [105,106]. As phosphoproteomics studies and indeed smaller, conventional studies continue to identify new phosphorylation sites on PKC…”