Phenotypic and Interaction Profiling of the Human Phosphatases Identifies
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Resource Phenotypic and Interaction Profiling of the Human Phosphatases Identifies Diverse Mitotic Regulators Graphical Abstract Authors Nicole St-Denis, Gagan D. Gupta, Zhen Yuan Lin, ..., Sally W.T. Cheung, Laurence Pelletier, Anne-Claude Gingras Correspondence [email protected] (L.P.), [email protected] (A.-C.G.) In Brief St-Denis et al. have systematically identified protein-protein interactions with phosphatases, enzymes important in cancer and other diseases. By combining this analysis with a functional screen, they uncover several tyrosine phosphatases with roles in cell division, providing potential targets for antimitotic cancer drugs. Highlights d The phosphatase interactome contains 1,335 interactions made by 140 phosphatases d Mitosis requires phosphatases of all evolutionary lineages, including several PTPs d PTPs play diverse roles in mitosis, notably in adhesion and centriole biogenesis d Combining complementary screens provides testable hypotheses about protein function St-Denis et al., 2016, Cell Reports 17, 2488–2501 November 22, 2016 ª 2016 The Author(s). http://dx.doi.org/10.1016/j.celrep.2016.10.078 Cell Reports Resource Phenotypic and Interaction Profiling of the Human Phosphatases Identifies Diverse Mitotic Regulators Nicole St-Denis,1 Gagan D. Gupta,1 Zhen Yuan Lin,1 Beatriz Gonzalez-Badillo,1,3 Amanda O. Veri,2 James D.R. Knight,1 Dushyandi Rajendran,1 Amber L. Couzens,1 Ko W. Currie,2 Johnny M. Tkach,1 Sally W.T. Cheung,1 Laurence Pelletier,1,2,* and Anne-Claude Gingras1,2,4,* 1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada 2Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada 3Present address: Batchelor Children’s Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA 4Lead Contact *Correspondence: [email protected] (L.P.), [email protected] (A.-C.G.) http://dx.doi.org/10.1016/j.celrep.2016.10.078 SUMMARY There are 150 genes in the human genome encoding proteins containing domains consistent with protein dephos- Reversible phosphorylation is a fundamental regula- phorylation (reviewed in Duan et al., 2015). This capability tory mechanism, intricately coordinated by kinases evolved independently on at least four occasions, resulting in and phosphatases, two classes of enzymes widely families with unique catalytic mechanisms and varied substrate disrupted in human disease. To better understand specificity (reviewed in Moorhead et al., 2009). Serine and thre- the functions of the relatively understudied phospha- onine residues are mainly dephosphorylated by phosphoprotein tases, we have used complementary affinity purifica- phosphatase (PPP) family phosphatases, including PP1 and PP2A, and metallo-dependent protein phosphatases (PPM, tion and proximity-based interaction proteomics also known as PP2C). These families diverge in sequence but approaches to generate a physical interactome for converge structurally at the catalytic center (Das et al., 1996). 140 human proteins harboring phosphatase catalytic Several members of the aspartate-based phosphatases (also domains. We identified 1,335 high-confidence inter- known as haloacid dehalogenases [HADs]) act on serine or tyro- actions (1,104 previously unreported), implicating sine phosphorylated proteins, although other family members these phosphatases in the regulation of a variety of preferentially dephosphorylate small molecules (reviewed in Sei- cellular processes. Systematic phenotypic profiling fried et al., 2013). The majority of tyrosine dephosphorylation, of phosphatase catalytic and regulatory subunits re- however, is mediated through the actions of the largest phos- vealed that phosphatases from every evolutionary phatase family—the protein tyrosine phosphatases (PTPs). family impinge on mitosis. Using clues from the inter- This family includes the receptor and nonreceptor PTPs and actome, we have uncovered unsuspected roles for the dual-specificity phosphatases (DSPs) that can dephosphor- ylate both tyrosine and serine/threonine residues, and in some DUSP19 in mitotic exit, CDC14A in regulating micro- cases, nonprotein substrates, including phospholipids (reviewed tubule integrity, PTPRF in mitotic retraction fiber in Alonso and Pulido, 2016). integrity, and DUSP23 in centriole duplication. The In this study, we employed two complementary proteomics functional phosphatase interactome further provides approaches to identify interaction partners for each human pro- a rich resource for ascribing functions for this impor- tein phosphatase, leading to an interactome of 1,335 interac- tant class of enzymes. tions (1,104 previously unreported) among 1,051 proteins. Although this alone provides a number of promising avenues for follow-up study, combining proteomics data with phenotypic INTRODUCTION screens can help place these interactions within specific biolog- ical contexts. We employed this strategy to examine mitosis—a Reversible protein phosphorylation, catalyzed by the reciprocal complex process involving widespread phosphoproteome reor- actions of kinases and phosphatases, is a ubiquitous signal ganization (Olsen et al., 2010). Several kinases are crucial for transduction mechanism, central to the regulation of many bio- mitosis and are being investigated as targets for clinical interven- logical processes. Of these two classes of enzymes, phospha- tion (Manchado et al., 2012). However, although much work has tases are comparatively poorly studied, despite their recognized been done on understanding the roles of a handful of phospha- involvement in diseases such as Alzheimer’s, diabetes, and can- tases in mitotic regulation (reviewed in Qian et al., 2013), the roles cer (reviewed in Braithwaite et al., 2012; Gurzov et al., 2015; and (if any) of most other phosphatases in mitotic progression are Julien et al., 2011, respectively). largely unknown. To address this, we herein complement our 2488 Cell Reports 17, 2488–2501, November 22, 2016 ª 2016 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). (legend on next page) Cell Reports 17, 2488–2501, November 22, 2016 2489 physical interaction screens by RNAi screening to identify and The PP1 and PP2A catalytic subunits, which together con- study phosphatases with roles in mitotic progression, leading tribute the bulk of serine/threonine phosphatase activity in the us to ascribe functions to several tyrosine family phosphatases cell (reviewed in Virshup and Shenolikar, 2009), displayed the in the regulation of mitosis. highest interactivity (>20 interactions each; Figure 1A). As ex- pected, a large fraction of the PP1 and PP2A interactomes RESULTS consisted of known regulatory subunits, which are thought to provide specificity to the dephosphorylation process (reviewed Interaction Networks Reveal a Role for Phosphatases in in Shi, 2009; Figure S2). Generally, PPP family members also a Variety of Cellular Processes generated more high-confidence interactions (mean of 18) than Many phosphatases are regulated though protein-protein inter- the average phosphatase (mean of 6). There was no correlation actions. For example, PPP family phosphatases such as PP1 between the relative abundance of the phosphatase bait (as de- and PP2A are regulated through combinatorial binding to a tected by spectral counts) and the number of high-confidence in- myriad of regulatory subunits (reviewed in Shi, 2009) and teractions (Figure 1A). MAPK-dephosphorylating DSPs dock to their substrates Recently, our laboratories have adopted a proximity-based through conserved sequences (reviewed in Peti and Page, method, BioID (Roux et al., 2012), as a complement to AP-MS. 2013). Interaction proteomics should thus be a useful starting BioID consists of the fusion of an abortive biotin ligase (BirA*) point for exploring the function of these enzymes. Here, we to a bait expressed in mammalian cells; addition of biotin cata- defined a protein phosphatase as any gene encoding one of lyzes its activation to biotinoyl-AMP by BirA* and allows covalent the four types of protein phosphatase catalytic domains labeling of lysines on proteins in the vicinity of the bait. Bio- (including inactive phosphatases), and compiled a list of 151 tinylated proteins are recovered by streptavidin affinity pull- genes of interest (Table S1; overlap with the DEPhOsphorylation down and identified by mass spectrometry. We previously Database [DEPOD] [Duan et al., 2015] is indicated). We gener- used BioID to detect phosphorylation-dependent interactions ated a library of Gateway entry clones and 3X-FLAG-tagged (Couzens et al., 2013), and for the identification of proximity destination vectors using the longest human isoform for each partners for components of relatively insoluble structures (Gupta phosphatase whenever possible to maximize the number of et al., 2015; Lambert et al., 2015). To survey the proximity putative interaction partners (see the Supplemental Experi- interaction landscape of phosphatases, and in particular of the mental Procedures). We next generated stable isogenic transmembrane receptor tyrosine phosphatases (RPTPs), we HEK293 Flp-In T-REx cell lines with tetracycline-inducible generated 60 stable cell lines with inducible expression of expression, resulting in the expression of 140 protein phospha- BirA*-tagged phosphatases, and performed BioID (Figure 1A; tases (Table S1; Figures S1A and S1B), 114 of which were pre- Table S3), revealing 2,358 proximity