Protein phosphatase 1 regulates the phosphorylation state of the polarity scaffold Par-3 Andreas Traweger*†, Giselle Wiggin*, Lorne Taylor*, Stephen A. Tate‡, Pavel Metalnikov*, and Tony Pawson*§¶ *Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; §Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; and ‡MDS Sciex, Concord, ON, Canada L4K 4V8 Contributed by Tony Pawson, May 2, 2008 (sent for review March 25, 2008) Phosphorylation of the polarity protein Par-3 by the serine/ key serine residues, thereby regulating its association with 14-3-3 threonine kinases aPKC␨/␫ and Par-1 (EMK1/MARK2) regulates proteins. Our data also suggest a role for PP1␣ in the formation of various aspects of epithelial cell polarity, but little is known about functional TJs by influencing the association of Par-3 and aPKC. the mechanisms by which these posttranslational modifications are reversed. We find that the serine/threonine protein phospha- Results tase PP1 (predominantly the ␣ isoform) binds Par-3, which localizes PP1␣ Is a Component of the Par-3 Complex and Localizes to Tight to tight junctions in MDCKII cells. PP1␣ can associate with multiple Junctions. To pursue the identities of Par-3-associated proteins, we sites on Par-3 while retaining its phosphatase activity. By using a used an affinity purification/mass spectrometry approach to iden- quantitative mass spectrometry-based technique, multiple reac- tify potential regulators of Par-3 function. Immunopurification of tion monitoring, we show that PP1␣ specifically dephosphorylates 3ϫFLAG-Par-3 from a stable 293T cell line followed by SDS/ Ser-144 and Ser-824 of mouse Par-3, as well as a peptide encom- PAGE and staining with colloidal Coomassie blue detected several passing Ser-885. Consistent with these observations, PP1␣ regu- proteins that coimmunoprecipitated with the fusion protein and lates the binding of 14-3-3 proteins and the atypical protein kinase that were not present in the control sample [supporting information C (aPKC) ␨ to Par-3. Furthermore, the induced expression of a (SI) Fig. S1A]. By using tandem mass spectrometry we identified catalytically inactive mutant of PP1␣ severely delays the formation several known interaction partners of Par-3 (Fig. S1B), including of functional tight junctions in MDCKII cells. Collectively, these Par-6, aPKC␫ (5), 14-3-3␧ (13, 14), angiomotin (19), and DNA-PK results show that Par-3 functions as a scaffold, coordinating both (20). In addition, analysis of proteins migrating at a relative mass of ␣ serine/threonine kinases and the PP1 phosphatase, thereby pro- 38,000 (Mr 38,000) revealed peptides from the catalytic subunit of viding dynamic control of the phosphorylation events that regu- the serine-threonine protein phosphatase PP1. We then immuno- late the Par-3/aPKC complex. precipitated endogenous Par-3 from a lysate prepared from mouse E13 embryonic tissue by using an antibody that detects the major epithelial polarity ͉ multiple reaction monitoring ͉ tight junctions ͉ Par-3 isoforms (180, 150, and 100 kDa) (8) and identified an phosphorylation dynamics association with endogenous PP1, but not with ␤-catenin used as a negative control (Fig. 1A). Similar results were obtained with 293T olarity is a common trait of eukaryotic cells and is critical for and MDCKII cells (data not shown). Furthermore, Par-3 did not Ptissue development and cellular diversity. Genetic and biochem- detectably coprecipitate the catalytic subunit of the serine/ ical studies of the ‘‘partitioning-defective’’ (par) phenotype in the threonine protein phosphatase PP2A (Fig. 1B). In addition, exog- Caenorhabditis elegans embryo, and of polarity defects in Drosophila enously expressed 2xmyc-tagged PP1 (isoform ␣) specifically bound melanogaster and mammalian epithelia and neuroblasts, have re- to yellow fluorescent protein (YFP) tagged Par-3 (100 kDa), but not vealed that cell polarization relies on the concerted activities of to YFP-␤-catenin or YFP alone (Fig. 1C). To assess the subcellular asymmetrically localized protein assemblies (1–3), one of which is distribution of PP1␣ and Par-3 we transfected MDCKII cells with the evolutionary conserved Par/atypical protein kinase C (aPKC) constructs encoding YFP-tagged Par-3 (180 kDa) and PP1␣ N- complex. The polarity protein Par-3, a functional component of this terminally tagged with a monomeric variant of red fluorescent complex, directly binds to the PDZ domain-containing protein protein (mRFP). As expected, fluorescence microscopy revealed Par-6 that, in turn, regulates the activity of associated aPKC by YFP-Par-3 at areas of cell–cell contact (10) (Fig. 1D Left). Addi- engaging the small GTPase Cdc42 (4, 5). However, Par-3 has also tionally, mRFP-PP1␣ strongly codistributed with YFP-Par-3 at the been implicated in various aspects of cell polarity independent of its plasma membrane and within cytoplasmic aggregates (Fig. 1D association with Par-6, including asymmetric cell division (6), Right). More importantly, spinning disk confocal microscopy re- dendritic and axonal development in neurons (7, 8), and directed vealed that endogenous PP1␣ codistributes with the tight junction cell migration (9). In mammalian epithelial cells Par-3 localizes to protein ZO-1 at the apical aspect of the plasma membrane (Fig. 1E the apical junctional complex (10) and is required for the estab- and Fig. S2A). lishment of apical-basal polarity and the formation of tight junctions The mammalian genome encodes four PP1 isoforms, PP1␣, (TJs) (11, 12). Many of the signaling events controlling cell polarity PP1␤, PP1␥1, and PP1␥2, with the latter two arising through and TJ formation are regulated by the activities of serine/threonine alternative splicing (21). Strikingly, probing endogenous Par-3 protein kinases, notably aPKC and Par-1 (EMK1/MARK2) (4–6, immunoprecipitates with isoform-specific antibodies revealed a 13–18). For example, aPKC maintains the apical membrane do- main by phosphorylating the basolateral determinants Lgl and Par-1, thereby releasing them from the cell cortex. Conversely, Author contributions: A.T. and T.P. designed research; A.T., G.W., L.T., and S.A.T. performed phosphorylation of Par-3 by Par-1 creates binding sites for 14-3-3 research; P.M. analyzed data; and A.T. and T.P. wrote the paper. proteins that, in turn, antagonize the association of Par-3 with The authors declare no conflict of interest. aPKC. Additionally, formation of an aPKC-Par-3 complex is neg- Freely available online through the PNAS open access option. atively regulated by aPKC phosphorylation of Par-3. In seeking †Present address: Baxter Innovations GmbH, 2304 Orth an der Donau, Austria. regulators that may counterbalance these phosphorylation events, ¶To whom correspondence should be addressed. E-mail: [email protected]. we identified the serine/threonine protein phosphatase 1 (PP1) as This article contains supporting information online at www.pnas.org/cgi/content/full/ a functional component of the Par-3 scaffold. We provide evidence 0804102105/DCSupplemental. that PP1␣ is required for the dephosphorylation of Par-3 at several © 2008 by The National Academy of Sciences of the USA 10402–10407 ͉ PNAS ͉ July 29, 2008 ͉ vol. 105 ͉ no. 30 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804102105 Downloaded by guest on September 25, 2021 pulldown assays by using purified regions of Par-3 fused to maltose- binding protein (MBP) and probed for their ability to bind purified PP1␣. In these experiments, the fragments spanning Par-3 PDZ1 and C-term2-1 (amino acid 1020–1217) showed a robust interaction with PP1␣, whereas PDZ3 bound more weakly and PDZ2, C-term2-2 (amino acid 1217–1333), or MBP alone did not bind (Fig. 2A). A common feature of many PP1-interacting proteins is the RVXF motif, [RK]-X0,1-[V/I]-[F/W], which mediates the associa- tion with a hydrophobic groove on the surface of PP1 (21). Par-3 harbors a single degenerate RVXF motif located at the C-terminal border of PDZ1, spanning residues 351–355 (RVIWF). Mutation of key residues to alanine (RVIWF to AVIWA) significantly decreased but did not completely abrogate binding of the first PDZ domain to PP1␣ (Fig. 2B). However, immunoprecipitating YFP-tagged Par-3 containing the RVXF mutation from 293T cells coexpressing 2xmyc PP1␣ did not reveal a substantial difference in binding when compared with wild-type YFP-Par-3, most likely because of the secondary binding sites present in Par-3 (Fig. S2C). Collectively, these results indicate that PP1␣ directly binds multiple regions of Par-3, including the first PDZ domain, which contains a degenerate RVXF motif. Par-3 Does Not Inhibit The Enzymatic Activity of PP1␣. To address whether Par-3 influences the activity of PP1␣, Par-3 immunocom- plexes (Fig. S4A), isolated from MDCKII cells, were monitored for their ability to dephosphorylate a phosphopeptide known to be a substrate for both PP1 and PP2A (Fig. 3A, DMSO). Low doses of okadaic acid (OA), which predominantly inhibit PP2A (23), had Fig. 1. Par-3 interacts with protein phosphatase 1. (A and B) Endogenous little effect on the Par-3-associated phosphatase activity, whereas ␤ Par-3 and -catenin immunoprecipitated from mouse E13 lysates were probed the addition of the PP1-specific inhibitor I-2 almost abolished the with anti PP1␣ or anti-PP2A antibodies. Preimmune (PI) serum was used as a control. (C) YFP immunoprecipitates (IPs) from MDCKII cells expressing 2xmyc- release of free phosphate (Fig. 3A). Subsequently, we tested PP1␣ and YFP-Par-3, YFP-␤-catenin, or YFP were immunoblotted (IB) with whether any of the PP1-binding regions of Par-3 had an impact on anti-myc (Top) or anti-GFP (Middle) antibodies. Whole-cell lysates (WCLs) were PP1 activity. As shown in Fig. 3B, 2xmyc-PP1␣ coimmunoprecipi- probed for equal expression of 2xmyc-PP1␣ by using an anti-myc antibody tating with either YFP-Par-3, YFP-PDZ1, YFP-PDZ3, or YFP-C- (Bottom).