Comprehensive Proteomic Analysis of Human Par Protein Complexes Reveals an Interconnected Protein Network
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m312171200
ISSN1083-351X
AutoresMiro Brajenovic, Gérard Joberty, Bernhard Küster, Tewis Bouwmeester, Gerard Drewes,
Tópico(s)Microtubule and mitosis dynamics
ResumoThe polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3ζ and η (Par-5), Par-6a, -b, -c, and PKCλ (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex “modules” formed around MARK4 (Par-1), Par-3·Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1·Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity. The polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3ζ and η (Par-5), Par-6a, -b, -c, and PKCλ (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex “modules” formed around MARK4 (Par-1), Par-3·Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1·Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity. Cell polarity is fundamental for the directional transport and positioning of vesicles and organelles, enabling the establishment of the embryonic body axis and cell fate decisions as well as maintaining fully functional differentiated cells (1Jan Y.N. Jan L.Y. Nature. 1998; 392: 775-778Crossref PubMed Scopus (240) Google Scholar, 2Mellman I. 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However their regulatory hierarchy seems to differ between organisms. In Drosophila, the localization of the PAR-3/PAR-6/PKC complex members is not co-dependent, nor are they, or PAR-5/14-3-3, required for the localization of PAR-1 during oogenesis (14Yamanaka T. Horikoshi Y. Suzuki A. Sugiyama Y. Kitamura K. Maniwa R. Nagai Y. Yamashita A. Hirose T. Ishikawa H. Ohno S. Genes Cells. 2001; 6: 721-731Crossref PubMed Scopus (246) Google Scholar, 15Benton R. Palacios I.M. St Johnston D. Dev Cell. 2002; 3: 659-671Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Later, in the oocyte, PAR-1 becomes dependent on PAR-3 (16Vaccari T. Ephrussi A. Curr. Biol. 2002; 12: 1524-1528Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). There is evidence that PAR-5/14-3-3 may also function downstream of PAR-1 (15Benton R. Palacios I.M. St Johnston D. Dev Cell. 2002; 3: 659-671Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Overexpression of Drosophila PAR-4/LKB1 partially rescues the par-1 phenotype, suggesting that the two kinases may function in a linear pathway (17Martin S.G. St Johnston D. Nature. 2003; 421: 379-384Crossref PubMed Scopus (258) Google Scholar). In mammals, Par-3 and Par-6 have been shown to control polarity in a variety of cell types. Par-6 forms the core of a polarity complex due to its direct interaction with Par-3, aPKC (λ or ζ), the GTPases Cdc42 or Rac, as well as with the tumor suppressor mLgl (18Qiu R.G. Abo A. Martin S.G. Curr. Biol. 2000; 10: 697-707Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 19Joberty G. Petersen C. Gao L. Macara I.G. Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (765) Google Scholar, 20Lin D. Edwards A.S. Fawcett J.P. Mbamalu G. Scott J.D. Pawson T. Nat. Cell Biol. 2000; 2: 540-547Crossref PubMed Scopus (47) Google Scholar, 21Plant P.J. Fawcett J.P. Lin D.C. Holdorf A.D. Binns K. Kulkarni S. Pawson T. Nat. Cell Biol. 2003; 5: 301-308Crossref PubMed Scopus (304) Google Scholar, 22Yamanaka T. Horikoshi Y. Sugiyama Y. Ishiyama C. Suzuki A. Hirose T. Iwamatsu A. Shinohara A. Ohno S. Curr. Biol. 2003; 13: 734-743Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). In epithelial cells, the binding of Par-3 and mLgl to Par-6·aPKC has been shown to be mutually exclusive resulting in opposite functions: Par-6 complexes containing Par-3 promote the formation of tight junctions, whereas complexes containing Lgl suppress them (21Plant P.J. Fawcett J.P. Lin D.C. Holdorf A.D. Binns K. Kulkarni S. Pawson T. Nat. Cell Biol. 2003; 5: 301-308Crossref PubMed Scopus (304) Google Scholar, 22Yamanaka T. Horikoshi Y. Sugiyama Y. Ishiyama C. Suzuki A. Hirose T. Iwamatsu A. Shinohara A. Ohno S. Curr. Biol. 2003; 13: 734-743Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 23Gao L. Joberty G. Macara I.G. Curr. Biol. 2002; 12: 221-225Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Par-6 may also be involved in the regulation of tight junctions by interacting with components of the crumbs complex (24Hurd T.W. Gao L. Roh M.H. Macara I.G. Margolis B. Nat. Cell Biol. 2003; 5: 137-142Crossref PubMed Scopus (410) Google Scholar). The role of the Par-6 complex has also been studied in other polarized cell types. In astrocytes, Par-6·aPKCζ is part of a pathway starting from integrins and the subsequent activation of Cdc42, leading to the control of migration. Both Par-6 and aPKC bind directly to glycogen synthase kinase 3β and subsequently regulate the binding of the adenomatous polyposis coli protein to microtubules leading to major reorganization of the cell, whereas Par-3 is not required (25Etienne-Manneville S. Hall A. Cell. 2001; 106: 489-498Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 26Etienne-Manneville S. Hall A. Nature. 2003; 421: 753-756Crossref PubMed Scopus (712) Google Scholar). However, in differentiating neurons, the Par-3·Par-6·aPKC complex is crucial for determining which neurite is selected to become the axon (27Shi S.H. Jan L.Y. Jan Y.N. Cell. 2003; 112: 63-75Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). The MARK kinases, which represent the mammalian homologs of PAR-1, were originally identified by their ability to phosphorylate microtubule-associated proteins (MAPs), and thus to regulate microtubule stability in cultured cells (28Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 29Drewes G. Trinczek B. Illenberger S. Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1995; 270: 7679-7688Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). MARKs were also shown to be necessary for the establishment of polarity in cultured epithelial and neuroblastoma cells (30Bohm H. Brinkmann V. Drab M. Henske A. Kurzchalia T.V. Curr. Biol. 1997; 7: 603-606Abstract Full Text Full Text PDF PubMed Google Scholar, 31Brown A.J. Hutchings C. Burke J.F. Mayne L.V. Mol. Cell Neurosci. 1999; 13: 119-130Crossref PubMed Scopus (26) Google Scholar). The human ortholog of PAR-4 is the serine/threonine kinase LKB1. Mutations in LKB1 cause Peutz-Jeghers syndrome, an autosomal-dominant disorder characterized by gastrointestinal polyps and an increased risk of neoplasms, indicating that LKB1 is a tumor suppressor (32Jenne D.E. Reimann H. Nezu J. Friedel W. Loff S. Jeschke R. Muller O. Back W. Zimmer M. Nat. Genet. 1998; 18: 38-43Crossref PubMed Scopus (984) Google Scholar). Failure to establish cell polarity may result in inappropriate overgrowth of differentiated cells and cause neoplasms (33Bardeesy N. Sinha M. Hezel A.F. Signoretti S. Hathaway N.A. Sharpless N.E. Loda M. Carrasco D.R. DePinho R.A. Nature. 2002; 419: 162-167Crossref PubMed Scopus (353) Google Scholar). Finally, the putative human orthologs of PAR-5, the 14-3-3 family proteins, have like LKB1 not been directly implied in cell polarity, but they are known to exert pleiotropic functions in processes crucial to polarity, e.g. vesicle trafficking (34O'Kelly I. Butler M.H. Zilberberg N. Goldstein S.A. Cell. 2002; 111: 577-588Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). It is not clear whether, in mammalian cells, the PAR proteins operate in a linear hierarchy similar to C. elegans and how they cooperate with other factors involved in polarity. Also it is not clear whether the orthologs of PAR-1 and PAR-4 operate in a protein complex similar to Par-3·Par-6 and how the “cross-talk” between the different polarity complexes is accomplished. In the present study, we employed tandem affinity purification (TAP) combined with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to map protein complexes around human PAR orthologs. We show that all PAR orthologs form protein complexes and that these complexes share common interactors. Our data allow the construction of a network connecting known and novel players in cell polarity and offer new insight into the molecular mechanisms of morphogenesis. cDNA Cloning—Expression vectors were generated by site-specific recombination (Gateway system, Invitrogen) of PCR-amplified ORFs into modified TAP-, HA-, or myc-tagged versions of the Moloney murine leukemia virus-based vector pZome1 (Cellzome; available from Euroscarf GmbH). For MARK4, LKB1, Par-6, aPKC, 14-3-3, and PNMA1, the TAP cassette was fused to the N terminus and for Par-3 to the C terminus. The inserted ORFs were subcloned from the following human MGC cDNA clones: aPKC, Image (National Institutes of Health): 4823853; 14-3-3ζ, Image: 2988020; 14-3-3η, Image: 3543571; LKB1, Image: 3689780; PNMA1, Image: 4431875; MARK4, Image: 3874322; MARK2, Image: 3139103; PAPK, Image: 3546243; CDC42 Image: 3626647; Mo25, Image: 4397573; Par-3, Image: 3939370; and Par-6C, Image: 4844008 (RZPD, Berlin, Germany). The Par-6a and b cDNAs were a kind gift of Dr. Ian Macara. Cell Culture—Human embryonic kidney cells (HEK293, ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Madin-Darby canine kidney (MDCK, ATCC) cells were maintained in minimal essential medium supplemented with 10% fetal calf serum and 2 mm glutamine. Pools of cells expressing epitope-tagged proteins were generated by retroviral gene transfer (35Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1900) Google Scholar). For transient expression, ORFs were fused N-terminally with HA- or myc-epitopes (36Jarvik J.W. Telmer C.A. Annu. Rev. Genet. 1998; 32: 601-618Crossref PubMed Scopus (147) Google Scholar). For microscopy, cells were grown on coverslips at 37 °C and 5% CO2 in a humidified chamber. Plasmids (300 ng) were transfected into cells grown at 70% confluency using lipofectamin2000 (Invitrogen) according to the manufacturer's instructions. Cells were fixed either with methanol for 10 min at -20 °C or with 4% (v/v) paraformaldehyde for 15 min at 37 °C. After fixation, cells were blocked with 1% bovine serum albumin/0.1% Triton X-100 in phosphate-buffered saline and incubated for 1 h at 37 °C with polyclonal anti-HA (Abcam, UK) or monoclonal anti-myc (BD Clontech). Secondary antibodies were goat anti-mouse- or anti-rabbit-labeled with Oregon green or Texas red. DNA was stained with 4′,6-diamidino-2-phenylindole. Coverslips were mounted on glass slides with Vectashield (Vector Laboratories), and cells were imaged using a ×60 objective on an Olympus IX70 inverted microscope. Immunoprecipitation—HEK293 cells were collected 48 h after transfection from one 10-cm dish in 400 μl of lysis buffer (250 mm Tris, pH 7.5, 25% glycerol, 7.5 mm MgCl2, 0.5% (v/v) Triton X-100, 500 mm NaCl, 125 mm NaF, 5 mm Na3VO4,1mm DTT, Roche Applied Science protease inhibitor tablets). Lysates were cleared by centrifugation and used for immunoprecipitation with anti-myc (BD Clontech) or anti-HA resins (Roche Diagnostics, Mannheim, Germany) for 2 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer and separated by SDS-PAGE. Proteins were transferred to nitrocellulose and detected using horseradish peroxidase-conjugated 3F10 or 9E10 at 1/10,000 (BD Clontech). For kinase assays, immunoprecipitates were washed three times with lysis buffer and once with kinase buffer (50 mm Hepes, pH 7.4, 10 mm MgCl2, 200 μm ATP, 100 mm NaCl, 1 mm DTT). Beads were incubated with 1 μCi of [γ-32P]ATP for 15 min at 30 °C, and the reaction was stopped with phosphoric acid. The samples were resolved on SDS-PAGE and/or spotted onto P81 paper, washed with 8% phosphoric acid, and analyzed by autoradiography. A specific peptide substrate, NVK-SKIGSTENLK was used as substrate for MARK (28Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Tandem Affinity Purification—Cell lysates were incubated with 200 μl of IgG-agarose beads (Sigma) for 2 h at 4 °C (37Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar). The beads were collected in 0.8-ml spin columns (MoBiTec) and washed with cleavage buffer (10 mm Tris, pH 7.5, 100 mm NaCl, 1 mm DTT, 0.1% Igepal, 0.5 mm EDTA). The beads were resuspended in cleavage buffer and incubated with 100 units of tobacco etch virus protease (Invitrogen) for 1 h at 16 °C. The eluate was transferred into a column containing 200 μl of calmodulin-agarose (Stratagene) in 10 mm Tris, pH 7.5, 100 mm NaCl, 1 mm DTT, 0.1% Igepal, 2 mm MgCl2, 2 mm imidazole, 4 mm CaCl2 and after washing with excess buffer the column was eluted with 600 μl of elution buffer (10 mm Tris, pH 8.0, 5 mm EGTA) at 37 °C. The freeze-dried eluates were separated on 4–12% NuPAGE gels (Novex) and stained with colloidal Coomassie. Mass Spectrometry—Gels were sliced into 1.25-mm bands across the entire separation range of each lane to sample all potential interacting proteins without bias with respect to size and relative abundance. Cut bands were reduced, alkylated, and digested as described previously (38Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7820) Google Scholar), and peptides were sequenced by tandem mass spectrometry (LC-MS/MS, QTOF Ultima and CapLC, Waters) (39Schirle M. Heurtier M.A. Kuster B. Mol. Cell Proteomics. 2003; 2: 1297-1305Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Proteins were identified by comparing the mass spectrometry sequence data against an in-house-curated version of the International Protein Index (available at www.ebi.ac.uk/IPI/) using the Mascot software package (Matrix Science, London, UK). Most proteins were unambiguously identified by the sequencing of several independent peptides (Supplemental Material, Table 1). For the few cases in which only one or two peptides supported the identification of a particular protein, we required these peptides to have a Mascot score indicating a <5% probability that the match could be considered a random event (40Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6776) Google Scholar). In addition, these peptide matches were confirmed by using expressed sequence tags as an alternative search algorithm (41Mann M. Wilm M. Anal. Chem. 1994; 66: 4390-4399Crossref PubMed Scopus (1317) Google Scholar). A set of “sticky” proteins typically identified in the majority of TAP purifications, as judged by filtering against a control dataset consisting of more than 400 TAP purifications, were excluded from the analysis. This set includes heat shock proteins, ribosomal proteins, keratins, actin, myosin, and α- and β-tubulins. All experiments were carried out in duplicates. Affinity Purification of Protein Complexes—Using retrovirus-mediated gene transfer, we created a panel of HEK293 cell pools expressing TAP-tagged fusions of nine human proteins representing putative orthologs of the C. elegans PAR gene products: MARK4 (C. elegans PAR-1), Par-3, LKB1 (C. elegans PAR-4), 14-3-3η, and ζ (C. elegans PAR-5), Par-6A, Par-6B, Par-6C, and PKCλ (C. elegans PKC3). TAP/MS analysis of the protein complexes formed around these nine proteins was performed from HEK293 cells, because we have assembled a data base that contains more than 5000 protein-protein interactions generated by TAP analysis in this cell line, which allows the reliable assessment of the specificity of a given interaction (data not shown). Expression levels were gauged by varying the multiplicity of infection and were typically 5–10 times over endogenous levels. Expanded cell pools were subjected to tandem affinity purification, a procedure consisting of two specific binding and two specific elution steps under mild conditions, which preserve the integrity of non-transient protein-protein interactions (37Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar). The affinity-purified complexes were resolved on SDS-PAGE and Coomassie-stained (Fig. 1). The tagged bait proteins represent the most prominent bands on the gel, and in all cases distinct copurifying proteins are present (Fig. 1). Proteins were identified by peptide sequencing using tandem mass spectrometry (LC-MS/MS). Most proteins were identified by the sequencing of several peptides (Table 1). Details on protein identification procedures are given under “Experimental Procedures.” As a representative example, Fig. 2 shows the identification of two interactors in the LKB1·Par-4 complex, which are identified by sequenced peptides covering 65% (Mo25) and 26% (PAPK) of their total sequence, respectively.Fig. 2Examples of protein identifications: Two novel interactors of LKB1·Par-4. A, LC-MS/MS spectrum of the tryptic peptide LLSAEFLEQHYDR from the Mo25 along with the underscored protein sequence showing all peptides that were sequenced from this protein. B, same for the tryptic peptide AVILSHFFR from PAPK.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TAP Analysis of Par-3·Par-6·aPKC Complexes—We subjected several core members of the Par-6 polarity complex to TAP/MS analysis (Par-6C, Par-3, and aPKCλ (Fig. 1); Par-6A, Par-6B, and Fig. S1 in Supplemental Material). This complex has been studied extensively by yeast two-hybrid and co-immunoprecipitation analysis (19Joberty G. Petersen C. Gao L. Macara I.G. Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (765) Google Scholar, 20Lin D. Edwards A.S. Fawcett J.P. Mbamalu G. Scott J.D. Pawson T. Nat. Cell Biol. 2000; 2: 540-547Crossref PubMed Scopus (47) Google Scholar), but to date a mapping of the polarity complex from mammalian cells by comprehensive tagging of the core scaffold proteins has not been reported. We detected the known components Par-6, Par-3, aPKC, and Lgl in all cases (Fig. 1). Cdc42 was detected only in the three TAP-Par-6 purifications and Rac was not found, presumably because the interaction of the small GTPases with Par-6 is transient. As previously reported, we found glycogen synthase kinase 3β to interact with all three Par-6 proteins, albeit only detectable by immunoblotting (data not shown) (26Etienne-Manneville S. Hall A. Nature. 2003; 421: 753-756Crossref PubMed Scopus (712) Google Scholar). Par-3 and Lgl are thought to bind competitively to the same site on Par-6 (21Plant P.J. Fawcett J.P. Lin D.C. Holdorf A.D. Binns K. Kulkarni S. Pawson T. Nat. Cell Biol. 2003; 5: 301-308Crossref PubMed Scopus (304) Google Scholar, 22Yamanaka T. Horikoshi Y. Sugiyama Y. Ishiyama C. Suzuki A. Hirose T. Iwamatsu A. Shinohara A. Ohno S. Curr. Biol. 2003; 13: 734-743Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Nonetheless low amounts of Lgl were detected, judged by low sequence coverage, in the TAP-Par-3 purification. Although it has been previously reported that Lgl2 does not bind to Par-6C (22Yamanaka T. Horikoshi Y. Sugiyama Y. Ishiyama C. Suzuki A. Hirose T. Iwamatsu A. Shinohara A. Ohno S. Curr. Biol. 2003; 13: 734-743Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), we found it to be present abundantly in TAP-Par-6C. Notably, each Par-6 paralog was also found to be in complex with the two other paralogs. Because they do not bind directly to each other (19Joberty G. Petersen C. Gao L. Macara I.G. Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (765) Google Scholar), this suggests the presence of higher order complexes. Several new interactors of the Par-6 polarity complex were identified (Table 1 and Figs. 1E and S1). Hensin, a large protein involved in the terminal differentiation of epithelia, was found in TAP-Par-6B (Fig. S1 in the Supplemental Material) and several proteins lacking functional annotation were found in TAP-Par-6C, including a protein related to Drosophila Spaghetti, a microfilament-binding protein involved in cytokinesis. In TAP-PKCλ, several novel interacting proteins were identified in addition to the known Sequestosome protein (42Sanz L. Sanchez P. Lallena M.J. Diaz-Meco M.T. Moscat J. EMBO J. 1999; 18: 3044-3053Crossref PubMed Scopus (328) Google Scholar) (see Fig. 1F and Table 1). However, because these interactors were not found by tagging of the other polarity complex members, it is likely that they are involved in PKCλ functions distinct from the control of polarity. Unexpectedly, we found MARK2, another homolog of C. elegans PAR-1, in the TAP-Par-6A purification, and its paralog MARK4 was identified with TAP-aPKCλ, suggesting molecular cross-talk between the Par-6 polarity complexes and MARK·Par-1 complexes. We found several more components shared between the different polarity complexes: 14-3-3 proteins, which represent homologs of C. elegans PAR-5, were found with TAP-Par-3, and PNMA1, a marker for paraneoplastic syndrome, was identified with TAP-Par-3, TAP-14-3-3, and TAP-MARK4 (Fig. 1), but not with any other protein in our HEK293 dataset (data not shown). Reciprocally, we identified Par-3 and PKCλ in a TAP-PNMA1 complex (Fig. S2 in the Supplemental Material). TAP Analysis of MARK4·Par-1 Complexes—The homologs of C. elegans PAR-1 are protein serine kinases conserved from yeast to man (28Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 43Drewes G. Nurse P. FEBS Lett. 2003; 554: 45-49Crossref PubMed Scopus (37) Google Scholar). The mammalian MARK·Par-1 family comprises four closely related gene products. In flies, PAR-1 has been reported to interact with 14-3-3 proteins (15Benton R. Palacios I.M. St Johnston D. Dev Cell. 2002; 3: 659-671Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This interaction is conserved, because we find several different 14-3-3 proteins in TAP-MARK4. 14-3-3 proteins are known to occur as homo- and heterodimers in cells and hence can serve as bridging factors. By co-immunoprecipitation it appears that MARK4 and MARK2 interact mainly with 14-3-3η, whereas Par-3 interacts mainly with 14-3-3ζ (see Fig. S1 in Supplementary Material). In total, the analysis of TAP-MARK4 resulted in the unambiguous identification of 20 interacting proteins (Fig. 1A). Among these, PKCλ, and cdc42 are components of the Par-6 polarity complex, and homologs of transforming growth factor-β-induced anti-apoptotic factor and 14-3-3 have been implicated in cell polarity. Transforming growth factor-β-induced anti-apoptotic factor may be an ortholog of Miranda, a protein that is associated with centrosomes and is involved in neuroblast asymmetric division in Drosophila (44Mollinari C. Lange B. Gonzalez C. Biol. Cell. 2002; 94: 1-13Crossref PubMed Scopus (18) Google Scholar). Interestingly MARK4 also associates with centrosomes and microtubules (45Trinczek B. Brajenovic M. Ebneth A. Drewes G. J. Biol. Chem. 2004; 279: 5915-5923Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Several interactors appear to be associated with the cytoskeleton; e.g. ARHGEF2, microtubule-associated exchange factor for Rac and Rho GTPases. We also find the microtubule-associated phosphatase 2A associated with TAP-MARK, which has been, like MARK·Par-1 itself, implicated in the regulation of the microtubule-associated protein tau (46Sontag E. Nunbhakdi-Craig V. Lee G. Brandt R. Kamibayashi C. Kuret J. White 3rd, C.L. Mumby M.C. Bloom G.S. J. Biol. Chem. 1999; 274: 25490-25498Abstract Full Text Full Text PDF PubMed Scopus (294) Google Sch
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