Quantitative Phosphoproteomics analysis based on [1]

Mitosis is characterized by the activation of mitotic kinases and inhibition of phosphoprotein phosphatases (PPPs), resulting in a stark increase in substrate phosphorylation that initiates mitotic progression. This balance of activities is reversed at the metaphase-to-anaphase transition. Besides the antagonistic action of protein kinases and phosphatases at the level of substrate phosphorylation, it is becoming apparent that reciprocal regulation of PPPs by protein kinases and vice-a-versa is an essential regulatory mechanism crucial for the generation of distinct cell cycle phases. While kinases have been extensively studied in this regard, we are just starting to elucidate PPP regulation. PPPs are responsible for the majority of serine/threonine dephosphorylation in eukaryotic cells and achieve substrate selectivity and specificity through the formation of holoenzyme complexes. Thus, the identification and quantification of PPP holoenzyme complexes in different cell cycle phases is important to elucidate their regulatory function. We have recently developed a mass spectrometry-based chemical proteomics approach for the enrichment, identification, and quantification of endogenous PPP holoenzyme complexes. Here, we combine PPP and kinase profiling to investigate the phosphorylation-dependent regulation of PPPs in mitosis. Comparison with global protein abundances is employed to determine the regulatory mechanism governing these changes. We demonstrate the utility of this approach by identifying that Cyclin-dependent kinase 1 (Cdk1) as the kinase that phosphorylates the catalytic subunit of PP2A (PP2Ac) on threonine 304. We show that this phosphorylation disrupts PP2A-B55 holoenzyme formation and thereby regulates mitotic entry and exit through alteration of substrate phosphorylation.

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A regulated phosphorylation sites are known. This hampers our understanding of its tumor suppressor functions and the mechanisms of site-specific dephosphorylation. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Dephosphorylation of ADAM17 decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways.

Mitosis is a process involving a complex series of events that require careful coordination. Protein phosphorylation by a small number of kinases, in particular Aurora A, Aurora B, the cyclin-dependent kinase-cyclin complex Cdk1/cyclinB, and Polo-like kinase 1 (Plk1), orchestrates almost every step of cell division, from entry into mitosis to cytokinesis. To discover more about the functions of Aurora A, Aurora B, and kinases of the Plk family, we mapped mitotic phosphorylation sites to these kinases through the combined use of quantitative phosphoproteomics and selective targeting of kinase activities by small-molecule inhibitors. Using this integrated approach, we connected 778 phosphorylation sites on 562 proteins with these enzymes in cells arrested in mitosis. By connecting the kinases to protein complexes, we associated these kinases with functional modules. In addition to predicting previously unknown functions, this work establishes additional substrate-recognition motifs for these kinases and provides an analytical template for further use in dissecting kinase signaling events in other areas of cellular signaling and systems biology.