Quantitative site-specific phosphorylation dynamics of human protein kinases during mitotic progression

Abstract

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.

Fig. 1.

Schematic overview of experimental strategy.

Fig. 2.

Outline and quality control of cell synchronization procedure. A, eukaryotic cell cycle with emphasis on mitotic stages. Centrosomes are shown as red dots, chromosomes are depicted in blue, and microtubules are displayed in green. The synchronization steps used are indicated in the diagram with inward and outward directed arrows marking the addition and release of drugs. T and N refer to thymidine and nocodazole, respectively. B, representative pictures from prometaphase, metaphase, and anaphase cells. The yellow arrow highlights a microtubule cluster, the pink arrow marks a mitotic spindle, and the white arrow points at a midbody. Scale bars represent 10 μm. C, synchronization efficiencies were plotted as counted from the images. D, Western blot analyses of the samples using various mitotic markers. P stands for prometaphase samples obtained from nocodazole block (Stage 1), M represents metaphase samples from a MG132 (Stage 2) block, T marks telophase (Stage 3) samples from MG132 release, and S stands for thymidine-blocked HeLa cells in S phase. The S phase stage is not part of the proteomics experiment but was included in the Western blot analyses as a reference. pHistone, phosphorylate Histone.

Fig. 3.

Quantitative dynamics of phosphokinome across mitosis. A, percentage of class I phosphorylation sites that are up-regulated (upward directed bars) above a ratio of 1.5 or down-regulated below a ratio of 0.66 (downward directed bars) between metaphase and prometaphase and telophase and metaphase, respectively. Percentages of the measured ratios between metaphase and prometaphase (B) and between telophase and metaphase (C) are depicted separately for phosphorylated kinase peptides (red) and unphosphorylated kinase peptides (blue).

Fig. 4.

Phosphorylation dynamics of key mitotic kinases. The regulation of phosphorylation sites (red triangle, up-regulated; green triangle, down-regulated; blue square, regulation below threshold; black circle, no data available) is shown for the ratios between prometaphase and S phase (first position; data taken from Daub et al. (31)), between metaphase and prometaphase (second position), and between telophase and metaphase. Data were taken from experiment 3 (best synchronization) if available. Phosphorylation sites marked with a star were only detected in experiment 1 or 2. AURK, Aurora kinase.

Fig. 5.

Interaction networks and enriched functional annotations. A, selected part of an interaction network extracted from a group of proteins with down-regulated phosphorylation sites centered on Cdk1; inset B shows the enrichment of Cdk1 interactors in the complete interaction network of proteins with down-regulated phosphorylation sites (supplemental Fig. S6) compared with proteins with up-regulated phosphorylation sites (supplemental Fig. S7) and a random group from the UniProt data set. C highlights that the functional annotation term “microtubule skeleton” is enriched in this network. D, selected part of an interaction network extracted from a group of proteins with up-regulated phosphorylation sites in telophase, containing many MAP kinases; inset E shows the enrichment of MAPK interactors in the complete interaction network of proteins with up-regulated phosphorylation sites (supplemental Fig. S7) compared with proteins with down-regulated phosphorylation sites (supplemental Fig. S6) and a random group from the UniProt data set. The enrichment of the functional annotation “nuclear membrane” in this network is shown in F. AURK, Aurora kinase.

Fig. 6.

Activation segment phosphorylation of MAP kinases in mitosis. A, dynamics of activation segment phosphorylation of different MAP kinases identified in the study. B, Western blots depicting the activation segment phosphorylation of p38 and ERK1/2 MAP kinases. T stands for thymidine-blocked HeLa cells in S phase. N represents the prometaphase sample obtained from a nocodazole block. Upon release from nocodazole, cells were harvested at six different time points. N* represents the sample for which the nocodazole block was continued for 150 min (′) after the initial nocodazole block. Phosphorylation on p38 and ERK1/2 is observed only in the cells that were released into telophase.

Fig. 7.

Correlation between kinase phosphorylation site regulation at mitotic entry and mitotic exit. The proportions of the class I kinase phosphorylation sites that were found up-regulated (red), not significantly changed (blue), or down-regulated (green) between S phase and prometaphase (the ratios were taken from Daub et al. (31)) in relation to their regulation in telophase are shown.