Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2011 Jun 28;4(179):rs5.
doi: 10.1126/scisignal.2001497.

Quantitative Phosphoproteomics Identifies Substrates and Functional Modules of Aurora and Polo-like Kinase Activities in Mitotic Cells

Affiliations
Free PMC article

Quantitative Phosphoproteomics Identifies Substrates and Functional Modules of Aurora and Polo-like Kinase Activities in Mitotic Cells

Arminja N Kettenbach et al. Sci Signal. .
Free PMC article

Abstract

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.

Figures

Fig. 1
Fig. 1
Characterization of small-molecule inhibitors of Aurora A, Aurora B, and Plks. (A) Western blot analysis of BI2536, MLN8054, AZD1152, and ZM447439 inhibitor titration in Taxol-arrested HeLa cells using antibodies against the phosphorylated T loop of all three Aurora kinases, the phosphorylated T loop of Plk1 (pT210), Cdc25c, and phosphorylated Ser10 of histone H3 (pS10). Anti–lamin A/C blots are shown as loading controls. Plk1 Thr210 is phosphorylated by Aurora A, Cdc25c is phosphorylated by Plk1, and his-tone H3 Ser10 is phosphorylated by Aurora B. Blot shown is representative of four independent experiments. (B) Raw LC-MS/MS data extracted for mass/charge ratios (m/z) corresponding to the Aurora A pT288 and Aurora B pT232 autophosphorylated activation loop phosphopeptides and the Plk1 pT210 activation loop phosphopeptide under each inhibitor condition. Blue trace: peptide from light, control cells; green trace: peptide from heavy, inhibitor-treated cells. All traces are extracted to ±2 parts per million (ppm) mass deviation from theoretical. Y axis is relative abundance on an arbitrary scale. (C) Immuno-fluorescence micrographs of spindles of control cells or cells treated with the indicated inhibitors. CREST (red) is a marker of kinetochores, and tubulin (green) marks microtubules. Proteins were labeled with antibodies. DNA (blue) was labeled with PicoGreen. Scale bar, 3 µm.
Fig. 2
Fig. 2
Cluster and motif analysis of putative Aurora A and B substrates. (A) Candidate Aurora A and B substrates identified by hierarchical clustering of ModSites with ratios reduced in either the MLN5 or the AZDZM conditions by 2.5-fold or more relative to uninhibited cells (na, not available). (B) Line graphs of log2 ratios of the ModSites of the corresponding Aurora A and B subclusters from panel A. The red line represents the average. (C) Motif analysis of both Aurora A–subclustered ModSites. (D). Motif analysis of all three Aurora B–subclustered ModSites.
Fig. 3
Fig. 3
Cluster and motif analysis of putative Plk substrates and regulation of Plk by Aurora A. (A) Candidate Plk substrates identified by hierarchical clustering of ModSites with ratios reduced by 2.5-fold or more in either the BI entry or the BI mitosis condition relative to uninhibited cells. (B) Motif analysis of all Plk phosphorylation sites from the array in panel A. (C) Averaged ratios of all Plk substrates shown in panel A. Error bars indicate 1 SD. The difference between MLN1 or MLN5 and AZDZM is significant as indicated by P values. (D) Motif analysis of ModSites in the −0.5 MLN5 ratio space. Plk consensus motifs are shown.
Fig. 4
Fig. 4
Evolutionary site versus motif conservation. Representative sequence alignments of one ModSite for the chromosome-associated kinesin KIF4A [O95239_(T799, S801, T803)] with indicated site (purple) and motif conservation (yellow and green). This ModSite is a predicted substrate of Aurora B. The combination of site and motif conservation of T799 indicates this as the site most likely phosphorylated by Aurora B relative to the other ambiguous sites in this identifier. Abbreviations for the amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; R, Arg; S, Ser; T, Thr; and Y, Tyr.
Fig. 5
Fig. 5
Candidate Aurora A, Aurora B, and Plk substrates and their spindle localization. (A) Diagram of proteins and unique ModSites identified in HeLa cells by quantitative chemical phosphoproteomics. Total number of identifications is indicated in black. Blue squares indicate candidate Plk substrates; green squares, candidate Aurora A substrates; and orange squares, candidate Aurora B substrates. Number of proteins and ModSites in each group that displayed >2.5-fold reduction in phosphorylation after inhibitor treatment, as well as those that contained a minimal kinase motif (in parentheses) is given. (B) Depiction of candidate Aurora A (green), Aurora B (orange), Aurora ambiguous (purple), and Plk (blue) substrates based on their subcellular localization to centromeres or kinetochores, centrosomes, and the spindle. Proteins with more than one colored letter are substrates of more than one of these kinases. The number of colored letters is not an indication of the number of phosphorylation sites matched to a particular kinase.
Fig. 6
Fig. 6
Verification of kinase substrates by in vitro kinase reactions (A). Scheme of phosphorylation site identification in cells and in vitro. (B) Coomassie gel of purified substrates for in vitro kinase reactions. (C) Reciprocal plots of MS2 peptide fragmentation spectra identified as part of the large-scale proteomics analysis and in the in vitro kinase reactions for the Aurora A substrate MAPRE3, the Aurora B substrate SPAG7, and the Plk1 substrate COPS8.
Fig. 7
Fig. 7
Identification of Plk1-interacting proteins. (A) Schematic of the mechanism of Plk1 activation and substrate recognition. Phosphorylation of Plk1 at Thr210 by kinases, such as Aurora A, leads to its activation. Through its Polo-box domain (PBD), Plk1 recognizes and interacts with some of its substrates, which have previously been phosphorylated by a priming kinase at an Sp[S/T]P sequence, leading to additional phosphorylation of these substrates by Plk1. PBD-independent interactions of Plk1 and substrates, which subsequently lead to phosphorylation of these substrates by Plk1, are also possible. (B) Scheme and Coomassie gel of Plk1 immunoprecipitation (IP). Plk1 and IgG bands are indicated by arrows. Substrates of Plk1 that interact directly with Plk1 through the PDB (labeled as A in panel A) or through other sites (labeled as C in panel A), as well as proteins bound to PBD-binding partners (secondary interactions, labeled as B in panel A) and direct Plk1-interacting proteins that are not substrates (not shown in panel A) are expected to coprecipitate. (C) Diagram of overlap of proteins and unique ModSites identified as Plk1 interactors by Plk1 IP (dotted black box) and Plk substrates identified in the large-scale phosphoproteomics experiments with BI2526 (solid blue box). (D) Distribution of [S/T]P and S[S/T]P sites in the whole proteome, in the 6061 proteins identified across all experiments (mitotic proteins), in all Plk1-interacting proteins identified in the Plk1 IP, in the 424 BI2536-sensitive candidate substrates, and in the 119 protein intersection of BI2536-sensitive substrates that were also in the Plk1 IP.
Fig. 8
Fig. 8
Analysis of NuMA phosphorylation. (A) Scheme of NuMA protein structure with domains, nuclear localization sequence (NLS), and identified phosphorylation sites. Mt-binding, microtubule binding. (B) Micrographs of endogenous NuMA (red) and tubulin (green) immunofluorescence in meta-phase in control and MLN8054-treated HeLa cells. (C) Micrographs of GFP–NuMA-WT, of GFP–NuMA-S1969A, and of GFP–NuMA-S1969E (red) and tubulin (green) immunofluorescence. (D) Depiction of Aurora A phosphorylation of NuMA on Ser1969 in the Mt-binding domain and its effect on NuMA spindle localization. (E). Micrographs of GFP–NuMA-S1969A (red) and tubulin (green) immunofluorescence in a multipolar spindle. Quantification of percentage of multipolar spindles in HeLa cells transfected with GFP–NuMA-WT, GFP–NuMA-S1969A, or GFP–NuMA-S1969E. The number of cells evaluated is indicated. Images in panels B, C, and E are maximum z projections of selected spinning disc confocal optical sections. Scale bar, 3 µm.

Similar articles

See all similar articles

Cited by 190 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback