Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits
2020; Springer Nature; Volume: 39; Issue: 13 Linguagem: Inglês
10.15252/embj.2019103695
ISSN1460-2075
AutoresThomas Kruse, Sebastian Gnosa, Isha Nasa, Dimitriya H. Garvanska, Jamin B. Hein, Hieu Nguyen, Jacob Samsøe‐Petersen, Blanca López‐Méndez, Emil Peter Thrane Hertz, Jeanette Schwarz, Hanna Sofia Pena, Denise Nikodemus, Marie Kveiborg, Arminja N. Kettenbach, Jakob Nilsson,
Tópico(s)Biochemical and Molecular Research
ResumoArticle13 May 2020Open Access Source DataTransparent process Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits Thomas Kruse Thomas Kruse orcid.org/0000-0002-2619-7388 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Sebastian Peter Gnosa Sebastian Peter Gnosa Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Isha Nasa Isha Nasa Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Cancer Center, Lebanon, NH, USA Search for more papers by this author Dimitriya Hristoforova Garvanska Dimitriya Hristoforova Garvanska Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jamin B Hein Jamin B Hein Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Hieu Nguyen Hieu Nguyen Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Cancer Center, Lebanon, NH, USA Search for more papers by this author Jacob Samsøe-Petersen Jacob Samsøe-Petersen Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Blanca Lopez-Mendez Blanca Lopez-Mendez Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Emil Peter Thrane Hertz Emil Peter Thrane Hertz Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jeanette Schwarz Jeanette Schwarz Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Hanna Sofia Pena Hanna Sofia Pena Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Denise Nikodemus Denise Nikodemus orcid.org/0000-0003-4492-817X Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Marie Kveiborg Corresponding Author Marie Kveiborg [email protected] orcid.org/0000-0002-1293-1019 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Arminja N Kettenbach Corresponding Author Arminja N Kettenbach [email protected] orcid.org/0000-0003-3979-4576 Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Cancer Center, Lebanon, NH, USA Search for more papers by this author Jakob Nilsson Corresponding Author Jakob Nilsson [email protected] orcid.org/0000-0003-4100-1125 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Thomas Kruse Thomas Kruse orcid.org/0000-0002-2619-7388 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Sebastian Peter Gnosa Sebastian Peter Gnosa Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Isha Nasa Isha Nasa Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Cancer Center, Lebanon, NH, USA Search for more papers by this author Dimitriya Hristoforova Garvanska Dimitriya Hristoforova Garvanska Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jamin B Hein Jamin B Hein Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Hieu Nguyen Hieu Nguyen Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Cancer Center, Lebanon, NH, USA Search for more papers by this author Jacob Samsøe-Petersen Jacob Samsøe-Petersen Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Blanca Lopez-Mendez Blanca Lopez-Mendez Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Emil Peter Thrane Hertz Emil Peter Thrane Hertz Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jeanette Schwarz Jeanette Schwarz Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Hanna Sofia Pena Hanna Sofia Pena Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Denise Nikodemus Denise Nikodemus orcid.org/0000-0003-4492-817X Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Marie Kveiborg Corresponding Author Marie Kveiborg [email protected]ric.ku.dk orcid.org/0000-0002-1293-1019 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Arminja N Kettenbach Corresponding Author Arminja N Kettenbach [email protected] orcid.org/0000-0003-3979-4576 Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Cancer Center, Lebanon, NH, USA Search for more papers by this author Jakob Nilsson Corresponding Author Jakob Nilsson [email protected] orcid.org/0000-0003-4100-1125 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Thomas Kruse1,‡, Sebastian Peter Gnosa2,‡, Isha Nasa3,4,‡, Dimitriya Hristoforova Garvanska1,‡, Jamin B Hein1, Hieu Nguyen3,4, Jacob Samsøe-Petersen2, Blanca Lopez-Mendez1, Emil Peter Thrane Hertz1, Jeanette Schwarz2, Hanna Sofia Pena2, Denise Nikodemus2, Marie Kveiborg *,2, Arminja N Kettenbach *,3,4 and Jakob Nilsson *,1 1Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark 3Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA 4Norris Cotton Cancer Center, Lebanon, NH, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +45 35325679; E-mail: [email protected] *Corresponding author. Tel: +1 603 653 9068; E-mail: [email protected] *Corresponding author. Tel: +45 21328025; E-mail: [email protected] The EMBO Journal (2020)39:e103695https://doi.org/10.15252/embj.2019103695 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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 the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. 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 B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease 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. Synopsis Distinct regulatory B-subunits convey differing specificities on the essential phosphatase PP2A. Phosphoproteomic substrate identification of two PP2A holoenzymes reveal that B-subunits directly affect dephosphorylation site preference of the common catalytic subunit. Phosphoproteomic strategies allow global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Regulatory B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit. Affinity and position of binding motifs modulate phosphorylation site selection of PP2A-B56. PP2A-B56 binding to the cancer target ADAM17 decreases growth factor signaling and tumor development in mice. Introduction It has been appreciated for over 40 years that multisite phosphorylation is a major mechanism for regulating protein function (Cohen, 2000). Historically, the differential phosphorylation of a protein was attributed to the actions of tightly regulated kinase activities. In this scenario, kinases are the major determinants of multisite phosphorylation with protein phosphatases being promiscuous counteractors. Kinases achieve specificity through a deep catalytic cleft that recognize specific amino acid consensus sequences flanking the phosphorylation site as well as through interactions with short linear motifs (SLiMs) in substrates (Miller & Turk, 2018). Our understanding of kinase function has been greatly facilitated by combining specific inhibitors with phosphoproteomics allowing global substrate identification. However, our understanding of phosphatase substrates is currently limited due to lack of specific inhibitors. Phosphoprotein phosphatase 2A (PP2A) accounts for the majority of phosphoserine and phosphothreonine dephosphorylation in eukaryotic cells and regulates many aspects of cellular physiology (Virshup & Shenolikar, 2009; Nilsson, 2019). PP2A is a heterotrimer composed of a catalytic C-subunit, a scaffolding A-subunit, and a regulatory B-subunit. The B-type subunits belong to four distinct gene families, B (B55), B′ (B56), B″ (PR72), and B‴ (PR93), each encoding two to five isoforms (Virshup & Shenolikar, 2009; Wlodarchak & Xing, 2016). The B55 and B56 families are the largest of the regulatory subunit families comprising four and five human isoforms, respectively. Recent discoveries have shown that PP2A similarly to kinases interacts with substrates and substrate specifying proteins through SLiMs (Cundell et al, 2016; Hertz et al, 2016; Wang et al, 2016; Wu et al, 2017). In the case of PP2A-B56, a series of biochemical and structural studies have revealed that proteins containing LxxIxE type of motifs engage a conserved binding pocket on all isoforms of B56 regulatory subunits (Hertz et al, 2016; Wang et al, 2016). For PP2A-B55, clusters of basic residues surrounding the phosphorylation site likely bind to a conserved acidic surface on the B55 subunit (Cundell et al, 2016). However, once bound to their substrates to which extent and how the PP2A holoenzymes selectively dephosphorylate individual sites is not known. One possibility is that substrate binding per se provides the proper three-dimensional positioning (key-in-lock model) of the PP2A active site for some phosphorylation sites but not others (Xu et al, 2006, 2008; Cho & Xu, 2007). Alternatively, it has been suggested that, like kinases, phosphatases may favor certain amino acid sequences immediately surrounding the phosphorylation site (Ubersax & Ferrell, 2007; Saraf et al, 2010; McCloy et al, 2015). However, only a small number of phosphorylation sites have been experimentally linked to specific PP2A holoenzymes making it difficult to conclude on general principles of phosphorylation site specificity. Furthermore, a direct comparison of substrates for two PP2A holoenzymes would be needed to determine whether regulatory subunits only act as targeting subunits or have additional roles in site-specific dephosphorylation. Uncovering this would have important implications for understanding how phosphorylation-mediated signaling is regulated. Results Development of a specific PP2A-B56 inhibitor To understand principles of PP2A specificity, we focused on PP2A-B56 which is a major tumor suppressor (Janssens et al, 2005; Eichhorn et al, 2009). Proteins containing LxxIxE motifs engage a conserved binding pocket on B56 regulatory subunits with varying micromolar affinities depending on the exact amino acid composition of the motif (Hertz et al, 2016; Wang et al, 2016; Wu et al, 2017). We recently showed that high-affinity LxxIxE motifs, when expressed in vivo, inhibit dephosphorylation by PP2A-B56 of the Ebola VP30 transcription factor (Kruse et al, 2018), probably by acting like a competitive inhibitor displacing PP2A-B56 from its substrate. Provided sufficient specificity and potency, we reasoned that such an inhibitor could be used to displace PP2A-B56 from all cellular LxxIxE containing interactors and, thus, to interrogate the phosphoproteome regulated by this phosphatase. To test this, we designed a series of constructs containing 1, 2, or 4 copies of a functional high-affinity LxxIxE motif separated by spacer sequences and fused these to either polyhistidine (His-tag) or yellow fluorescent protein (YFP) (Fig 1A). Constructs containing 4 copies of a non-binding AxxAxA motif were included as controls. The His-tagged inhibitor series were expressed and purified from Escherichia coli, and their binding to recombinant B56α was analyzed using isothermal titration calorimetry (ITC). Indeed, both the binding affinities (KD) and the stoichiometry (number of B56 molecules bound per inhibitor) increased with the number of LxxIxE motifs (Figs 1A and B, and EV1A). Prolonged mitosis is a well-established mitotic phenotype of interfering with PP2A-B56 function (Foley et al, 2011; Suijkerbuijk et al, 2012; Kruse et al, 2013). To assess the potency of the inhibitor series in cells, the YFP-tagged constructs were transfected into HeLa cells and progression through mitosis was monitored by live-cell microscopy. A clear correlation between phenotype severity and inhibitor copy number was observed (Fig 1C). Based on these results, we focused on the YFP-tagged 4x(LxxIxE) B56 inhibitor and the corresponding 4x(AxxAxA) control inhibitor. To determine the specificity of the B56 inhibitor, it was affinity-purified from HeLa cells and interacting proteins were identified by quantitative label-free mass spectrometry (MS). Strikingly, all components of the PP2A-B56 holoenzyme were strongly enriched in elutes from B56 inhibitor samples compared to control inhibitor samples (Fig 1D and Table EV1). This includes the five isoforms of B56 regulatory subunits and the two isoforms of each of the catalytic and scaffold subunits. NSF (N-ethylmaleimide-sensitive fusion protein), a vesicle-fusing ATPase, was the only other protein that bound specifically to the B56 inhibitor. Thus, the B56 inhibitor displays excellent specificity toward the PP2A-B56 holoenzyme family. We also concluded from this experiment that most proteins directly interacting with PP2A-B56 engage the LxxIxE binding pocket for effective binding. Figure 1. Development of a PP2A-B56 specific inhibitor A, B. Schematic of the B56 inhibitor series and affinities and stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection, and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results. C. Time from nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analyzed. Median time is indicated by red line. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann–Whitney U-test was used for statistical analysis (ns: non-significant, ***P ≤ 0.001). D. Volcano plot representing mass spectrometry identified proteins co-purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A-B56 subunits co-purifying with the B56 inhibitor are indicated. E, F. Competition assay in HeLa cells stably expressing RFP-tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP-B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink—B56 SLiM-containing protein and known B56 interactor; blue—known B56 interactor, green—B56 SLiM-containing protein) (E) or Western blotting (F). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Binding affinity of B56 inhibitors to recombinant B56 ITC measurements using B56α and indicated His-tag versions of ctrl and B56 inhibitors. Download figure Download PowerPoint Next, we tested whether the B56 inhibitor is able to displace PP2A-B56 interactors. To this end, YFP-B56α was transfected into HeLa cells stably expressing mCherry-tagged B56 inhibitor or control inhibitor. Purifications of YFP-B56α were subsequently analyzed by quantitative label-free MS (Fig 1E and Table EV2) or Western blotting (WB) (Fig 1F) probing with antibodies against Separase, Kif4A, BubR1, and Axin1, which all contain validated LxxIxE motifs (Hertz et al, 2016). Both MS and WB analyses revealed that YFP-B56α enriched from cells expressing the B56 inhibitor shows a significant decrease in interactor binding compared to YFP-B56α enriched from cells expressing the control inhibitor. In summary, we have developed a competitive inhibitor with excellent potency and specificity toward PP2A-B56. Identification of PP2A-dependent phosphorylation sites We next used this inhibitor to identify in vivo substrates of PP2A-B56. Stable HeLa cell lines allowing rapid induction of the B56 or control inhibitor were synchronized at either G1/S or in mitosis (M), and cells were collected and processed for quantitative phosphoproteomics analysis (Figs 2A and B, and EV2A and B). Using this approach, we identified and quantified a total of 13,515 and 27,745 phosphorylation sites in G1/S and M, respectively (Fig 2A and B, Table EV3). Of these sites, 548 and 398 were significantly increased in phosphorylation upon B56 versus control inhibitor expression (log2 ratio > 0.8 (1.75-fold), P-value < 0.05, phosphorylation site localization probability > 75%) in G1/S and M, respectively. The phosphorylation sites that increased were located on 651 proteins of which 34 contain a validated or predicted LxxIxE motif (Hertz et al, 2016; Wu et al, 2017). It was previously shown that LxxIxE containing proteins can act as scaffolds for the recruitment of other proteins for dephosphorylation (Suijkerbuijk et al, 2012; Qian et al, 2017; Kruse et al, 2018). Using the STRING database (Jensen et al, 2009), we found that an additional 491 of the 651 proteins identified in our screen are direct interactors of proteins with validated or predicted LxxIxE docking motifs. Thus, the majority of up-regulated phosphorylation sites are present on LxxIxE containing proteins or on their immediate interactors strongly supporting the notion that the identified phosphorylation sites are PP2A-B56 targets. Figure 2. Phosphorylation site preference of PP2A-B56 A, B. Schematic of synchronization protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light gray. Phosphorylation sites in a protein containing an LxxIxE motif are colored pink. C–E. IceLogo representation of over- and underrepresented amino acid residues surrounding phosphorylation sites for the indicated experiments (letter coloring is standard iceLogo color output). F, G. IceLogo representation of over- and underrepresented amino acid residues surrounding phosphorylation sites for the experiments indicated in Fig EV2F and G, respectively (letter coloring is standard iceLogo color output). H. Distribution of phosphorylation site consensus motifs surrounding B56- or B55-dependent up-regulated phosphorylation sites (top panel) in comparison with all phosphorylation sites (bottom panel) in the indicated experiment (proline-directed: pS/TP; basic: R/KxxpS/T, R/KxpS/T, R/KpS/T; acidic: D/E/NxpS/T, D/EpS/T, pS/TD/E, pS/TxD/E; x—any amino acid). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Phosphoproteomics on cells expressing the B56 inhibitor Live-cell imaging of cells expressing doxycycline-inducible B56 inhibitor. Cell were released from a thymidine block and followed into mitosis. Doxycycline was added at time 0 h. Quantification of mitotic duration of cells from (A) Each circle represents a single cell and median time in mitosis indicated by red line. A representative result from at least three independent experiments is shown. At least 20 cells were counted per condition in the experiment shown. NEBD; nuclear envelope breakdown. Comparison of log2 ratios of B56-dependent dephosphorylation sites versus other phosphorylation sites on the same protein. IceLogo representation of over- and underrepresented amino acid residues surrounding phosphorylation sites for the down-regulated phosphorylation sites from experiments presented in Fig 2A and B. Venn diagram showing overlap of PP2A-B56-regulated sites in G1/S and mitosis (M). Schematic of in vitro peptide phosphorylation assay set-up corresponding to IceLogo of PP2A-B56-regulated sites in Fig 2F. Schematic of LxxIxE inhibition experiment in mitotic lysate corresponding to IceLogo of PP2A-B56-regulated sites in Fig 2G. Mitotic cell lysates treated with thiophosphorylated wt or S62A GST-Arrp19 for 5 min followed by Western blotting with the antibodies indicated. Source data are available online for this figure. Download figure Download PowerPoint In general, dephosphorylation of a site was a specific event that did not affect all phosphorylation sites on a protein. Comparison of log2 ratios of B56-dependent dephosphorylation sites versus other phosphorylation sites on the same protein was not correlated (R = 0.1113; Fig EV2C). To explore the mechanism for this differential site specificity, the chemical nature of the phosphorylation sites dephosphorylated by PP2A-B56 was investigated. Over- and underrepresentation of amino acids surrounding the up-regulated phosphorylation site were determined by comparison with all phosphorylation sites identified in the respective screens (Colaert et al, 2009). This revealed a preference for basic amino acids upstream of the dephosphorylation site (Fig 2C and D). Interestingly, we found a strong deselection for phosphorylation sites containing a proline residue in the +1 position. In contrast, no preference pattern was observed when we analyzed the 184 and 163 phosphorylation sites that were significantly decreased in phosphorylation upon B56 versus control inhibitor expression (log2 ratio > 0.8 (1.75-fold), P-value < 0.05, phosphorylation site localization probability > 75%) in G1/S and M, respectively (Fig EV2D). Furthermore, among all increased phosphorylation sites co-identified in both G1/S and M datasets, the overlap of PP2A-B56-regulated sites was only 4% (Fig EV2E). This indicates unique B56 substrates in mitosis and G1/S, yet the datasets reveal remarkably similar phosphorylation site consensus motif preferences. The phosphorylation site preference of PP2A-B56 was further investigated by two independent experimental methods. First, we performed an in vitro peptide phosphatase assay that sampled phosphopeptides purified from cells. Purified PP2A-B56α holoenzyme was added and dephosphorylation kinetics followed by mass spectrometry (Figs 2F and EV2F, Table EV4). Second, we added an LxxIxE inhibitor peptide or a control peptide to cell extracts and determined inhibition of dephosphorylation after 5 and 15 min (Figs 2G and EV2G, Table EV5). Both approaches confirmed the apparent preference for basophilic and a deselection of proline-directed phosphorylation sites. Moreover, neither of the PP2A-B56 phosphoproteomics screens revealed a preference of this phosphatase for phosphorylated threonines over phosphorylated serines. These findings are in stark contrast to previous observations that proline-directed phosphorylation sites are excellent substrates of PP2A-B55, another major PP2A holoenzyme species, and that this phosphatase shows a clear preference for phosphothreonine over phosphoserine (Agostinis et al, 1992; Cundell et al, 2016; Godfrey et al, 2017). To investigate this difference further, we identified PP2A-B55-regulated phosphorylation sites by adding thiophosphorylated Arpp19, a potent mitotic PP2A-B55 inhibitor (Gharbi-Ayachi et al, 2010; Mochida et al, 2010), or thiophosphorylated Arpp19 S62A as a control to mitotic cell extracts and compared these samples quantitatively by mass spectrometry or Western blotting with α-pTP antibodies (Fig EV2H). This identified 1405 PP2A-B55 up-regulated sites (log2 ratio > 0.8 (1.75-fold), P-value < 0.05, phosphorylation site localization probability > 75%) of which less than 1.3% was shared with the sites identified in the PP2A-B56 datasets. Moreover, these results confirmed the differential preference of PP2A-B55 and PP2A-B56 for proline-directed motifs and the previously reported preference of PP2A-B55 for phosphothreonine over phosphoserine (Fig 2E and Table EV6). Comparison of all increased phosphorylation sites in the PP2A-B56 G1/S and M as well as the PP2A-B55 datasets supports the notion that while basophilic, acidophilic, and proline-directed sites can be dephosphorylated by the two phosphatase holoenzymes, PP2A-B56 and PP2A-B55 show a clear difference in their relative preferences (Fig 2H). Differential phosphorylation site preference of PP2A-B56 and PP2A-B55 Next, we wanted to investigate whether the observed dephosphorylation site patterns are inherent properties of the two PP2A holoenzymes. In principle, preference of certain phosphorylation sites as observed in the phosphoproteomics analysis could be biased by PP2A holoenzymes localizing to specific subcellular compartments enriched for certain kinases. First, we established an in vitro phosphatase assay with purified PP2A-B56α and PP2A-B55α holoenzymes using synthetic phosphopeptides with phosphorylation sites surrounded by amino acids conforming to physiologically relevant basophilic (PKC), acidophilic (PLK1), or proline-directed (CDK1) kinase consensus sequences (Fig 3A). Kinetic analysis revealed that the Michaelis–Menten constant Km was roughly similar for PP2A-B56 and PP2A-B55 toward the three different phosphopeptides. On the other hand, the catalytic efficiency (Kcat/Km) of PP2A-B56 toward the proline-directed phosphopeptide was 50- to 100-fold lower compared to the acidophilic and basophilic ones, whereas this difference was not observed for the PP2A-B55 holoenzyme. Figure 3. Differential phosphorylation site preference of PP2A-B56 and PP2A-B55 A. Michaelis–Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phosphopeptides. Mean and standard deviation shown in plots as black bars (n = 3 independent experiments). B–D. In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n = 3 independent experiments). Download figure Download PowerPoint The PP2A-B55 phosphoproteomics experiment (Fig 2E) predicts a preference for basic amino acids and a deselection of acidic residues C-terminal to the phosphorylated TP sites. Deselection of basic amino acid residues was observed N-terminal to the TP sites. A panel of synthetic peptides with a variable content of amino acid residues flanking the phosphorylated TP site almost completely recapitulated this in vitro (Fig 3B). The PP2A-B56 iceLogos predict a preference for basic amino acids N-terminal to the phosphorylation site and a deselection of prolines in the +1 position, whereas no phosphothreonine over phosphoserine preference was observed (Fig 2C, D, F and G). A series of phosphopeptides designed to test this confirmed that PP2A-B56 shows reduced dephosphorylation of phosphorylation sites with a proline in the +1 position (Fig 3C). However, the preference for basic amino acids N-terminal to
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