Systematic phosphorylation analysis of human mitotic protein complexes

Abstract

Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.

Fig. 1. Overview of the phosphorylation analysis of the mitotic interaction network

(A) Schematic outline of the workflow for mitotic gene selection, BAC tagging, phosphoprotein complex purification, and analysis. (B) Summary table of data obtained from the phosphorylation analysis of the mitotic interaction dataset.

Fig. 2. Identification of phosphosites in proteins from the mitotic interaction network

(A) Frequency distribution graph of the number of phosphosites identified per interaction cluster in the mitotic interaction dataset. (B) Interaction map for a selection of 9 out of 107 small clusters as determined in (14), showing the phosphorylation-site density in percent for each protein. Complexes containing reciprocal interactions are enclosed by solid gray lines; those without reciprocal interactions are denoted by dashed gray lines. Interactions between complexes are indicated by dashed blue lines with the number of interactions represented by line thickness. SFCM is the name of the clustering method. (C) Percent kinase motifs found within 615 high confidence hits found in 1451 phosphorylation sites. Note that 39% of all hits correspond to the mitotic kinases CDK1 (represented by three motif variants named Cdc2, CDK1-1, and CDK1-2), PLK1, AURKA, AURKB, and NEK2.

Fig. 3. Experimental conditions for the identification of mitosis-specific and kinase inhibitor-sensitive phosphorylation sites

(A) Western blots of cell extracts from the four conditions described in panel (C) probed with the indicated antibodies (representative of 2 experiments). (B) Histone H1 kinase activities of cell extracts from the conditions described in panel (C). Assays were performed on 4 samples per condition (N=4), error bars represent standard deviation, p-values [determined by Student’s t-test (77)] of ≤ 0.05 (*) or ≤ 0.005 (**) are indicated. (C) Scheme of the workflow for purification of phosphoprotein complexes and data analysis of four conditions: LOG = interphase, NOC = 18 hours nocodazole arrest, BI = 18 hours nocodazole arrest with additional treatment of 250 nM BI 4834 for last two hours, Hesp = 18 hours nocodazole arrest with additional treatment of 100 nM Hesperadin and 10 μM MG132 for last two hours. (D) Summary table of data obtained from phosphorylation analysis of 24 baits from samples as described in (C). (E) Percent kinase motifs found within 455 high confidence hits found in 618 phosphorylation sites. Note that 33% of all hits correspond to the mitotic kinases CDK1 (represented by three motif variants named Cdc2, CDK1-1, and CDK1-2), PLK1, AURKA, AURKB, and NEK2.

Fig. 4. Identification of phosphorylation sites in protein complexes from the four-condition dataset

(A) Silver-stained SDS-PAGE gel showing proteins purified from the four cell-treatment conditions (LOG, NOC, BI, Hesp) following a LAP-TAP purification using mouse Bub1b as bait. This gel illustrates the level of purity achieved in purifications carried out with 24 diferent baits. Annotated protein bands are based on their expected migration positions, the intensity of the marker bands corresponds to the annotated protein amounts in ng. (B) Phosphoproteins and phosphosites identified by MS analysis of the three protein complexes MCC, APC/C, and MIS12/NDC80 and the copurifying protein UBR5, the first column (c) indicates complex membership, affinity purified using the mouse Bub1b-LAP bait, from the four conditions, as shown in (A). The percentages results from the sequence coverages obtained after combining peptides generated by all proteases. (C) Interaction map for a selection of 8 out of 12 complexes analysed in the four treatment conditions, projected onto the interaction clusters determined in Hutchins et al. (14), showing the phosphorylation site density (in percent) for each protein and indicating proteins with sites dependent on PLK1 and AURKB.

Fig. 5. Phosphospecific antibodies validate MS results

(A) Phosphospecific antibodies (listed in panel E) were tested on cell extract (XT) or immunopurified protein (IP). XT were from interphase (LOG), noc-arrest (NOC), and noc-arrest in combination with 2 hours of BI 4834 (BI) or a combination of Hesperadin and MG132 (Hesp). The STAG2 pSer¹²⁶¹ antibody was tested on XT from synchronised cells harvested either in G2, in mitosis by shake-off (SO), in mitosis with 3 hours nocodazole treatment (sN), or in mitosis with 3 hours nocodazole and last 2 hours either in BI 4834 (sBI) or in Hesperadin plus MG132 (sHesp). Cell cycle stage was verified using the indicated antibodies. N(experiments)=2. (B) STAG2 and WAPAL were immunopurified from LOG and incubated with PLK1, ATP, and 1 μM of BI 2536. Western blots were performed using indicated antibodies. N(experiments)=2. (C) Immunofluorescence images of cells at different cell-cycle stages treated or not with 100 nM of BI 2536 for 30 min prior to fixation were stained with indicated antibodies and DAPI. N=3. (D) Phosphospecific antibodies were tested on XT or IP using the indicated antibodies or LAP purifications from indicated BAC-cells. N(experiments)=2. (E) Phosphospecific antibodies and antigenic peptides (#= ID number). The phosphosite category as determinded by MS and Western blotting (WB) is: i/m = detected in LOG and NOC, m = only in NOC, unphosphorylated in LOG, bi = unphosphorylated in BI, hes = unphosphorylated in Hesp).