The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle

Abstract

Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004457, 1-18, 2011.During mitosis, multiple processes, such as mitotic entry, spindle assembly, chromosome segregation, and cytokinesis, must be carefully coordinated to ensure the error-free distribution of chromosomes into the newly forming daughter cells. The physical separation of the chromosomes to opposite poles of the cell is driven by the mitotic spindle, a proteinaceous and highly dynamic microtubule (MT) 1 -based macromolecular machine. Spindle assembly begins early in mitosis and is completed when the bipolar attachment of microtubules to kinetochore (KT) pairs is achieved (1, 2). Polo-like kinase 1 (Plk1), a serine/threonine-specific kinase first identified in Drosophila (3), is one of the key regulators of this essential mitotic process and has therefore attracted much attention (4 -6). In agreement with its diverse functions, the localization of Plk1 during mitosis is dynamic. Plk1 first associates with centrosomes in prophase before it localizes to spindle poles and KTs in prometaphase and metaphase. During anaphase, Plk1 is recruited to the central spindle and finally accumulates at the midbody during telophase. Proteomics studies using oriented peptide libraries have shown that two so-called polo boxes at the C-terminal end of Plk1, the polo box domain (PBD), are crucial for the localization of this kinase to cellular structures (7,8). This domain binds to specific phosphorylated sequence motifs that are created by other priming kinases or are self-primed by Plk1 itself, thus providing an efficient mechanism to regulate localization and substrate selectivity in time and space (9 -11). Despite the pleiotropic and critical functions of Plk1 during mitosis, only a limited number of target proteins and phosphorylation sites on substrates have so far been identified or studied in detail...

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

“…5A) (9). Downstream targets of Plk1 were also prominent among constituents of the nuclear pore, which has recently been appreciated as a major cellular site of Plk1-mediated phosphoregulation (13). Notably, we found Plk1 regulation for 7-29% and identified phosphopeptides for 42-74% of all proteins annotated to the above mentioned and other cellular components enriched for Plk1-regulated phosphoproteins (supplemental Fig.…”

“…Moreover, a recent phosphoproteomics screen reported Plk1 substrate candidates in the mitotic spindle. In this study, phosphorylation changes were measured upon prolonged Plk1 inhibition by either small interference RNA expression or small molecule inhibitor treatment (13). However, none of the previous studies has systematically interrogated the effect of short term Plk1 modulation on a proteome-wide scale in a specificity-controlled manner.…”

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

“…S5). Recently, asparagine in the -2 position was also detected by a peptide library-based approach to find PLK1 motifs (42) and in a phospho-proteomic approach by Santamaria et al (38) supporting the hypothesis this motif represent a new variant of the PLK1 consensus site. Our bioinformatic analysis thus identified two potential variations of the published PLK1 consensus motif (43).…”

“…However, we cannot exclude that our approach failed to detect some PLK1-regulated phosphorylation sites, either because the corresponding phosphopeptides were not recovered, or because the 15 min BI 4834 treatment was not sufficient to allow dephosphorylation of phosphorylation sites that had been generated by PLK1. Very recently, another phospho-proteomic study identified 866 PLK1-dependent phosphorylation sites on 304 mitotic spindle proteins (38). We identified only 25 of these 866 phosphorylation sites.…”

TAK-960, a Novel, Orally Available, Selective Inhibitor of Polo-Like Kinase 1, Shows Broad-spectrum Preclinical Antitumor Activity in Multiple Dosing Regimens

“…Consistent with in vitro observations, TAK-960 treatment in mice bearing HT-29 colorectal cancer or K562ADR leukemia xenografts induced pHH3 in tumors and showed significant antitumor efficacy in vivo. Recently, a number of PLK1 substrates have been reported (25,26). Although the availability of an appropriate antibody against PLK1 substrates for immunohistochemistry or ELISA is still limited, the reliability of PLK1 substrates as potential predictive or diagnostic biomarkers should be compared with pHH3 in future studies.…”