Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate
2007; Springer Nature; Volume: 26; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601683
ISSN1460-2075
AutoresDrew M. Lowery, Karl R. Clauser, Majbrit Hjerrild, Dan Lim, Jes Alexander, Kazuhiro Kishi, Shao‐En Ong, Steen Gammeltoft, Steven A. Carr, Michael B. Yaffe,
Tópico(s)Fungal and yeast genetics research
ResumoArticle19 April 2007free access Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate Drew M Lowery Drew M Lowery Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Karl R Clauser Karl R Clauser Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Majbrit Hjerrild Majbrit Hjerrild Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark Search for more papers by this author Dan Lim Dan Lim Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Jes Alexander Jes Alexander Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Kazuhiro Kishi Kazuhiro Kishi Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Shao-En Ong Shao-En Ong Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Steen Gammeltoft Steen Gammeltoft Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark Search for more papers by this author Steven A Carr Corresponding Author Steven A Carr Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Michael B Yaffe Corresponding Author Michael B Yaffe Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Drew M Lowery Drew M Lowery Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Karl R Clauser Karl R Clauser Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Majbrit Hjerrild Majbrit Hjerrild Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark Search for more papers by this author Dan Lim Dan Lim Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Jes Alexander Jes Alexander Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Kazuhiro Kishi Kazuhiro Kishi Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Shao-En Ong Shao-En Ong Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Steen Gammeltoft Steen Gammeltoft Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark Search for more papers by this author Steven A Carr Corresponding Author Steven A Carr Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Michael B Yaffe Corresponding Author Michael B Yaffe Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Broad Institute of MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Author Information Drew M Lowery1,‡, Karl R Clauser2,‡, Majbrit Hjerrild1,3,‡, Dan Lim1, Jes Alexander1, Kazuhiro Kishi1, Shao-En Ong2, Steen Gammeltoft3, Steven A Carr 2 and Michael B Yaffe 1,2 1Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA 2Broad Institute of MIT and Harvard, Cambridge, MA, USA 3Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark ‡These authors contributed equally to this work *Corresponding authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge MA 02142, USA. E-mail: [email protected] MB Yaffe, Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Building E18-580, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Tel.: +1 617 452 2103; Fax: +1 617 452 4978; E-mail: [email protected] The EMBO Journal (2007)26:2262-2273https://doi.org/10.1038/sj.emboj.7601683 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine–threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled–coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression. Introduction In eukaryotic cells, Polo-like kinase-1 (Plk1) and related orthologues perform a wide variety of essential functions during mitosis (Barr et al, 2004; Glover, 2005; van de Weerdt and Medema, 2006). Levels of Plk1 peak in late G2 and early M, accompanied by dramatic changes in subcellular localization as cells transit through various mitotic stages (Golsteyn et al, 1994). During interphase and early prophase, Plk1 resides primarily at the centrosome, where it facilitates centrosome maturation, separation, and microtubule nucleation during late prophase and prometaphase (Lane and Nigg, 1996; Rapley et al, 2005; De Luca et al, 2006). By metaphase, a fraction of Plk1 has relocalized to the kinetochores, where it seems to be involved in regulating aspects of spindle checkpoint function and the metaphase–anaphase transition (Ahonen et al, 2005; Goto et al, 2006). During anaphase, Plk1 is concentrated in the spindle midzone, where it probably facilitates microtubule sliding, while following chromosome segregation, Plk1 remains in the central spindle and midbody, where it participates in ingression of the cleavage furrow (Neef et al, 2003; Liu et al, 2004). Particularly prominent cytokinetic phenotypes are observed in budding and fission yeast, where mutations in the respective Plk1 orthologues, Cdc5 and Plo1, result in incomplete assembly of actomyosin and septin ring structures along with delayed and improper deposition of septal material (Lee et al, 2005; Yoshida et al, 2006). Although these and related mutational studies have provided many insights into Cdc5/Plo1 function in simple model organisms, the diversity of Plk1 functions in higher eukaryotes makes it difficult to comprehensively identify Plk1-regulated pathways or define the bulk of the Plk1 interactome using standard molecular genetics techniques. Separation of function alleles are hard to identify owing to the presence of a single common binding pocket and substrate phosphorylation cleft shared by most, if not all substrates (Cheng et al, 2003; Elia et al, 2003b). Full genetic disruption of the Drosophila Plk1 orthologue, polo, causes embryonic lethality (Donaldson et al, 2001), whereas full depletion of the Xenopus Plk1 orthologue, Plx1, prevents mitotic entry (Qian et al, 2001). More recently, RNA interference has been used to examine the effect of Plk1 depletion in human cell lines, revealing a marked dependence of phenotype on the particular genetic background of the cell. In tumor-derived cell lines, depletion of Plk1 causes apoptosis along with mitotic catastrophe, making the delineation of specific Plk1 functions difficult (Spankuch-Schmitt et al, 2002; Liu et al, 2006). In other immortalized cell lines, depletion of Plk1 causes a delay in mitotic entry with subsequent arrest at prometaphase, preventing analysis of later phenotypes without sensitization strategies to avoid activation of various mitotic checkpoints. If both the DNA damage and spindle checkpoints are bypassed, Plk1-depleted cells can complete an apparently normal mitosis; however, chromosome segregation fails (van Vugt et al, 2004a, 2004b). In non-transformed cell lines, depletion of Plk1 causes only very minor cell-cycle defects, although co-depletion of p53 mimics the cell death phenotypes seen in tumor-derived cell lines (Liu et al, 2006). The various Plk1-depletion phenotypes are complicated by varying degrees of Plk1 knockdown, as a 90% knockdown of Plk1 in HeLa cells can allow normal mitotic processes whereas an ∼100% depletion completely blocks cell-cycle progression (Liu et al, 2005, 2006). In an effort to more comprehensively elucidate the substrates and interacting proteins of Plk1, we pursued a biochemical/proteomic approach. All Plks have a similar protein architecture including an N-terminal Ser/Thr kinase domain and a conserved C-terminal Polo-box domain (PBD) (Figure 1A). The PBD of Plks was previously identified in our lab as a pSer/pThr-binding module that specifically recognizes Ser-[pSer/pThr]-Pro/X motifs on peptides with low micromolar affinity (Elia et al, 2003a). Phospho-dependent ligand recognition by the PBD is necessary for the targeting of Plk1 to specific substrates (i.e. processive phosphorylation), as well as for the dynamic re-localization of Plk1 to specific subcellular structures during mitosis where other Plk1 targets reside (i.e. distributive phosphorylation) (Lowery et al, 2005). We therefore performed affinity purification and mass spectrometry studies to identify Plk1 PBD-interacting proteins from U2OS cells at different stages of the cell cycle. Figure 1.The PBD of Plk1 preferentially binds ligands in mitosis. (A) Domain structure of Plk1. Residues His-538 and Lys-540 are required for phosphopeptide binding by the PBD. (B) Experimental strategy for identifying cell-cycle-dependent PBD ligands. (C) Purity and equivalence of recombinant wild-type (WT) and mutant (MUT) PBDs used for interaction screening. Samples were analyzed by SDS–PAGE and stained with Coomassie blue. (D) U2OS cells were synchronized at the G1/S transition by a double thymidine block or in M-phase by nocodazole treatment, and DNA content analyzed by flow cytometry. (E) WT and MUT PBD were used to pull down interaction partners from double thymidine blocked or the nocodazole-arrested U2OS cell lysates. Bound proteins were separated by SDS–PAGE and visualized by SYPRO Ruby staining. Lines on the right of gel indicate where gel was cut before subsequent mass spectrometry analysis with each labeled section correlated with Supplementary Table S1. Download figure Download PowerPoint Our strategy was to define all putative interacting partners, and then select one particular sub-network to investigate in more detail at the molecular level. We identified approximately 600 proteins that are members of phosphorylation-dependent Plk1 PBD interacting complexes. These proteins are known to be involved in a wide variety of mitotic processes, including processes not previously thought to be regulated by Plk1. We chose to focus on the roles of Plk1 in regulating cytokinesis. The requirement for Plk1 and its orthologues for proper initiation and completion of cytokinesis has been well established in unicellular organisms (Ohkura et al, 1995; Song and Lee, 2001) and Drosophila (Carmena et al, 1998), and several previously described PBD-associated proteins are known to play roles in this process in mammalian cells (Neef et al, 2003; Zhou et al, 2003; Liu et al, 2004), although exactly how Plk1 fits into the complete cytokinesis network is unclear. One of the major upstream regulators of cytokinesis is RhoA, which, together with its downstream targets, controls the formation and constriction of the actomyosin ring at the cleavage furrow (Glotzer, 2005). The functions of Plk1 and Rho GTPases may be linked, as cytokinesis-specific GEFs for Rho were recently identified as Plk targets both in mammalian cells (Niiya et al, 2006) and by our recent work in budding yeast (Yoshida et al, 2006). We now report that Plk1 functions in multiple parallel overlapping pathways with RhoA, and directly interacts with a subset of critical effectors of RhoA. We demonstrate that Plk1 and RhoA synergize to maximally activate the cytokinetic protein kinase Rock2 in vitro and within cells, and that Plk1 and Rock2 interact directly in vivo in a phosphorylation-dependent mitosis-specific manner, with maximal colocalization at the midbody during cytokinesis. Results and discussion The Plk1 PBD shows mitosis-specific interactions with many proteins involved in diverse aspects of cell division To identify potential targets of Plk1, we examined the ability of the isolated PBD to bind to proteins in a cell cycle-dependent manner. Recombinant Plk1 PBD was expressed in bacteria and purified to homogeneity (Figure 1C). We also purified a His-538 Ala/Lys-540 Met double mutant form of the PBD, which does not bind to phosphorylated ligands (Elia et al, 2003b) and therefore serves as an optimal negative control to reveal sticky nonspecific interactions or phospho-independent protein–PBD interactions that might arise from high abundance. The wild-type and mutant PBD proteins were crosslinked to Sepharose CN-4B beads and used as an affinity matrix. Human osteosarcoma U2OS cells were arrested at the G1/S transition by a double thymidine block or arrested in mitosis by treatment with nocodazole. Cell-cycle synchronization was verified by FACS (Figure 1D). Lysates from these two cell populations were prepared and equal amounts of total protein were applied to columns containing either the wild-type or mutant PBD column. After extensive washing with a near neutral-pH medium-salt buffer that is not expected to disrupt complexes, PBD-interacting proteins were eluted off the columns by competition with an optimal PBD-binding phospho-peptide (YMQS-pT-PK) (Elia et al, 2003a). The recovered proteins were then separated by SDS–PAGE and visualized by SYPRO Ruby staining (Figure 1B, top). Both the wild-type and mutant PBD bound very weakly, and with similar affinity, to a variety of proteins in the G1/S cell lysate. In marked contrast, the wild-type PBD, but not the mutant PBD, showed very strong binding to a large number of proteins in the mitotic cell lysates (Figure 1E). The darkest band at 25 kDa is the PBD itself, indicating that there is some leeching of the PBD from the column. These observations suggest that the Plk1 PBD can specifically interact with many mitotic proteins in a phospho-dependent manner. Furthermore, because Plk1 has been reported to interact with microtubules (Feng et al, 1999), we used nocodazole treatment to obtain mitotically arrested cells, as this drug depolymerizes microtubules and should therefore minimize potential indirect interactions of the PBD with other microtubule-interacting proteins. The affinity-based purification assay shown in Figure 1E could either isolate mitotic proteins that bound directly to the PBD, or proteins that were not themselves direct PBD interactors but instead were components of larger PBD-associated complexes. The specificity of PBD binding was therefore investigated by Far-Western blotting. Many of the mitotic proteins captured with the wild-type PBD were capable of direct interaction. In contrast, none of the proteins that were retained by the mutant PBD showed a strong detectable interaction with the wild-type PBD in this assay (Figure 2A). Figure 2.Mitotic proteins bind to the PBD in a phospho-dependent manner and are substrates of Plk1. (A) Direct binding of PBD-interacting proteins. Proteins interacting with the wild-type (WT) and mutant (MUT) PBD from nocodazole-arrested U2OS cell lysates (Figure 1B) were transferred to PVDF membrane and analyzed for direct binding by Far-Western analysis using the WT PBD as a probe. (B) Phosphorylation-dependent PBD interactions. Nocodazole-arrested U2OS cell lysates were incubated with (+) or without (−) λ-protein phosphatase before WT-PBD pull-down. The bound proteins were separated by SDS–PAGE and visualized by SYPRO Ruby staining. (C) Plk1 phosphorylation of PBD-interacting proteins. Wild-type PBD was used to pull down interacting proteins from nocodazole-arrested U2OS cell lysates. The proteins were incubated with or without active Plk1 and [γ-32P]ATP, separated by SDS–PAGE, and visualized by autoradiography. Download figure Download PowerPoint To examine the extent to which the binding of mitotic ligands to the PBD is dependent upon phosphorylation, nocodazole-arrested cell lysates were dephosphorylated with λ-phosphatase before the PBD pull-down. As shown in Figure 2B, both the number of recovered ligands and intensity of ligand binding were greatly reduced after phosphatase treatment of the mitotic cell lysates. Next, to investigate if any of the Plk1 PBD-interacting proteins were also potential Plk1 substrates, the affinity-purified proteins were incubated with a constitutively active mutant of full-length Plk1, Plk1-T210D, in the presence of [γ-32P]ATP. As shown in Figure 2C, incubation with Plk1-T210D resulted in the direct phosphorylation of many of these PBD-interacting proteins. Taken together, the data in Figures 1 and 2 strongly support a model where the PBD facilitates the interaction of Plk1 with a wide range of mitotic targets that have undergone prior priming phosphorylation during mitosis, and indicates that a subset of the interacting proteins are themselves Plk1 substrates. Liquid chromatography tandem mass spectrometry (LC/MS/MS) was used to identify the mitotic PBD-interacting proteins. Each lane of the gel from the nocodazole-arrested cell lysates in Figure 1E was excised, cut into 12 pieces as indicated (Figure 1E), and subjected to in-gel digestion with trypsin. The extracted peptide mixtures were then separated using reverse phase HPLC, which was coupled to an LTQ-FT hybrid ion trap Fourier transform mass spectrometer for peptide identification (Figure 1B, bottom), and the corresponding proteins identified by database searching. For each protein, the relative ratio of wild-type/mutant PBD-bound abundances was determined using the sum of the extracted ion current measured for each sequenced peptide precursor ion in the intervening MS scans of the LC/MS/MS chromatogram. Proteins were then categorized as being wild-type specific (peptide ions present only in the wild-type PBD eluents), wild-type enriched (peptide ions present at >20-fold intensity in the wild-type PBD eluents compared to the mutant PBD eluents), and nonspecific (peptide ions present at ⩽20-fold intensity in the wild-type versus mutant PBD eluents). In total, we identified 622 distinct proteins that were at least 20-fold more abundant in the wild-type PBD pull-down compared to the mutant H538A/K540 M PBD pull-down and were considered to be potential PBD interaction partners (Supplementary Table S1). All proteins were characterized according to GO categories defining their molecular function (GO Consortium, 2001) (Figure 3A). Figure 3.The PBD interactome. (A) Only wild-type (WT) PBD-specific or -enriched interacting proteins that contain at least one match to the optimal PBD-binding motif (S-[S/T]-P) are shown ((B), blue boxes, column 2). Proteins were categorized according to their known biological function by GO terms. Blue text indicates those proteins in which we were able to map at least one S-[pS/pT]-P site; dark blue indicates those that also contain an optimal Plkl phosphorylation site, whereas light blue indicates those that do not. For the remaining proteins, those shown in purple contain an optimal Plk1 (E/D)X(S/T) φ phosphorylation site, whereas those shown in black do not. Previously known Plk1 interactors are indicated with an asterisk. (B) Summary statistics of proteins identified by mass spectrometry from Plk1 PBD pull-down assays in nocodazole-arrested U2OS cells. Proteins were divided into three categories based on specificity for the WT versus mutant (MUT) PBD. Dark red denotes proteins that bound only to the WT PBD; light red, proteins with ⩾20-fold enhanced binding to WT PBD; dark gray, proteins with <20-fold enhanced binding to WT PBD. The presence of optimal PBD-binding S-[S/T]-P motifs is shown by dark and light blue and gray filled bars in the second column. White boxes in the second column correspond to identified interacting proteins that do not contain the optimal PBD-binding motif. Potential Plk1 (E/D)X(S/T)φ phosphorylation sites are indicated by purple and gray wedges in pie charts. (C) A subset of phosphorylation sites in the WT specific/enriched versus nonspecific PBD-interacting proteins were mapped by mass spectrometry. Mapped sites were categorized into those that matched the optimal PBD-binding motif (S-[pS/pT]-P), the minimal peptide PBD-binding motif (S-[pS/pT]), the minimal cyclin-dependent kinase phosphorylation motif ([pS/pT]-P), or none of the above ([pS/pT]). Download figure Download PowerPoint We selected a small random subset of proteins identified in the mass spectrometry-based screen for further validation based on the availability of antibodies: Lamin A, Cofilin, MCMs, the protein kinase CK2 alpha, myosin phosphatase targeting subunit 1 (MYPT), and Rock2. Pull-downs from mitotic lysates followed by immunoblotting confirmed that all these proteins preferentially interacted with wild-type PBD compared to the mutant PBD (Supplementary Figure S1A). The cell-cycle-dependent interaction of the selected proteins with the PBD was investigated by performing in vitro pull-down assays with lysates from double thymidine blocked (G1) and nocodazole-treated (M) cells. As shown in Supplementary Figure S1B, interactions between the PBD and all of these proteins were mitotic specific. To investigate the requirement for phosphorylation, mitotic lysates were treated with λ-protein phosphatase before PBD pull-downs and immunoblotting. In each case, interaction of these proteins with the PBD was either eliminated or substantially reduced following phosphatase treatment (Supplementary Figure S1C). Thus, all six of the interactions tested were mitotic phosphorylation-dependent interactions. The complete set of proteins identified in our PBD interactome included proteins previously demonstrated to associate with Plk1 such as MCMs (Tsvetkov and Stern, 2005), septins (Song and Lee, 2001), anillin (shown to be a Plkl substrate in vitro; Straight et al, 2005), and members of the 20S proteasome complex (Feng et al, 2001). Some other known endogenous Plk1-interacting proteins, such as Cdc25C, were not identified in this screen, likely as a result of our nocodazole treatment/spindle checkpoint arrest strategy, which appeared to enrich for late mitotic targets. Plk1 targets such as Cdc25C and Wee1 are thought to play a role in entry into mitosis, and thus might not bind to Plk1 once mitosis is underway. Additional Plk1 interactors, such as Bub3, are only expected to be engaged after the spindle checkpoint is extinguished. Most of the PBD-interacting proteins identified in this study have not been previously reported. Many of those proteins participate in a broad range of cellular functions that show distinct changes during mitosis, transcription, translation, splicing, and metabolism (Figure 3A). Intriguingly, although protein synthesis is necessary for mitotic entry and progression, the overall rate of protein synthesis in mitotic cells has been reported to be markedly decreased to 25–30% of the rate seen in interphase cells (Tarnowka and Baglioni, 1979). At the same time, the synthesis of a number of proteins including c-myc is enhanced during mitosis (Kim et al, 2003). Transcription and mRNA splicing has also been shown to be inhibited during mitosis (Shin and Manley, 2002). The metabolic state of mitotic cells also undergoes significant alterations in order to accommodate disruption and distribution of membrane compartments and components (Warren, 1993). Recently, a transcriptional coactivator protein, Ndd1, has been discovered to be a direct substrate of Cdc5, the yeast Plk1 orthologue. Cdc5 was recruited to specific promoters where it phosphorylated Ndd1 to activate transcription of cell-cycle-regulated genes involved in mitotic progression (Darieva et al, 2006). Plk1 has also been shown to regulate the nuclear translocation of the transcription factor HSF1 (Kim et al, 2005). Thus, we anticipate further elucidation of the role of Plk1 in these processes. Bioinformatic analysis reveals that Plk1 PBD ligands are enriched in Ser-[Ser/Thr]-Pro motifs and are potential Plk1 substrates In order to help distinguish between indirect and direct interactors, each of the PBD-interacting proteins identified by mass spectrometry was evaluated for the presence of an optimal PBD recognition motif. Isolated phosphopeptides bind to the Plk1 PBD through the optimal consensus motif S-[pS/pT]-[P/X], where the Ser residue preceding the pSer/pThr makes three hydrogen-bonding interactions with the PBD (one through a bound water molecule) and contributes significantly to ligand affinity (Elia et al, 2003b). As seen in Figure 3B, PBD recognition motifs were identified in 47.3% of the wild-type-specific PBD-interacting proteins and 41.3% of the wild-type-enriched interactions. In contrast, only 16.2% of the nonspecific PBD-interacting proteins contained this motif. To determine whether these values were statistically significant, we generated 2000 'mock' protein data sets by randomly selecting either 622 (the total number of combined wild-type PBD-specific or -enriched interactions; Figure 3B, column 1, red boxes) or 277 proteins (the total number of nonspecific interactions; Figure 3B, column 1, gray box) from the current human NCBI RefSeq collection, and examined these data sets of randomly selected proteins for the percentage of proteins containing S-[S/T]-P motifs. As shown in Supplementary Figure S2A and B, the distribution of the number of protein in each of the random data sets containing S-[S/T]-P motifs was roughly normally distributed, with the same average value of 31.4%. The percentage of PBD-specific or -enriched interacting proteins containing S-[S/T]-P motifs that we identified in our mass spectrometry-based screen (average value 45.3%) was over 7 s.d. above the mean from that expected for a similar sized collection of random proteins (Supplementary Figure S2B). Likewise, the percentage of PBD nonspecific interacting proteins containing S-[S/T]-P motifs was over 5 s.d. below that expected (Supplementary Figure S2A). Thus, our non-biased method for identifying PBD interactors greatly enriched for proteins that contained the optimal motifs necessary for PBD binding, suggesting that the optimal PBD-binding motif for isolated phosphopeptides likely functions in an analogous way for many full-length phosphoprotein ligands. The wild-type PBD-specific or -enriched interacting proteins were also evaluated for the presence of potential Plk1 phosphorylation sites [E/D]-X-[S/T]-[F/L/I/Y/W/V/M] (Nakajima et al, 2003). Among the 282 proteins that contain a PBD recognition motif, this motif was found in 88% of the proteins. In contrast, among the 340 proteins that do not contain a PBD recognition motif, this phosphorylation motif was present in 75% of the proteins (Figure 3B). We performed a similar statistical analysis for putative Plk1 phosphorylation sites in random protein data sets containing either 282 proteins (the number of wild-type PBD-specific or -enriched interactions containing S-[S/T]-P motifs; Figure 3B, blue boxes, column 2) or 340 proteins (the number of wild-type PBD-specific or -enriched interactions not containing S-[S/T]-P motifs; Figure 3B, white boxes, column 2). This revealed a similar mean value of 74.4% (Supplementary Figure S2C and D). The co-occurrence of a Plk1 phosphorylation motif in the S-[S/T]-P motif-containing PBD-specific or -enriched proteins found in our mass spectrometry-based screen was nearly 5 s.d. greater than that expected by chance alone in a randomly selected set of proteins (Supplementary Figure S2D). In contrast, for the PBD-specific or -enriched proteins found in our mass spectrometry-base
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