Abstract
Protein phosphatase 2A (PP2A) is a highly abundant heterotrimeric Ser/Thr phosphatase involved in the regulation of a variety of signaling pathways. The PP2A phosphatase activator (PTPA) is an ATP-dependent activation chaperone, which plays a key role in the biogenesis of active PP2A. The C-terminal tail of the catalytic subunit of PP2A is highly conserved and can undergo a number of posttranslational modifications that serve to regulate the function of PP2A. Here we have studied structurally the interaction of PTPA with the conserved C-terminal tail of the catalytic subunit carrying different posttranslational modifications. We have identified an additional interaction site for the invariant C-terminal tail of the catalytic subunit on PTPA, which can be modulated via posttranslational modifications. We show that phosphorylation of Tyr307PP2A-C or carboxymethylation of Leu309PP2A-C abrogates or diminishes binding of the C-terminal tail, whereas phosphorylation of Thr304PP2A-C is of no consequence. We suggest that the invariant C-terminal residues of the catalytic subunit can act as affinity enhancer for different PP2A interaction partners, including PTPA, and a different ‘code’ of posttranslational modifications can favour interactions to one subunit over others.
Acknowledgments
We thank our group members for suggestions and comments on the manuscript. E.M.Q. was supported by The Danish Council for Independent Research (Medical Sciences; grant 271-09-0187). C.L. was supported by a European Molecular Biology Organization (EMBO) postdoctoral fellowship. This research was further supported by grants from the Swedish Research Council, Swedish Cancer Society, as well as a Singapore NRF-CRP (National Research Foundation – Competitive Research Programme) grant. We thank Diamond Light Source for access to beamline I24 (MX5873 and MX6603) that contributed to the results presented here. We acknowledge technical support by the SPC facility at EMBL Hamburg and the Protein Science Facility at the Karolinska Institutet for providing crystallization infrastructure. The research leading to these results has furthermore received funding from the European Community’s Seventh Framework Program (FP7/2007–2013) under BioStruct-X (grant agreement N°783). M.K. and J.B. were supported by the DFG (GRK1026), the BMBF (ProNet-T3), and ERDF by the EU.
References
Cayla, X., Van Hoof, C., Bosch, M., Waelkens, E., Vandekerckhove, J., Peeters, B., Merlevede, W., and Goris, J. (1994). Molecular cloning, expression, and characterization of PTPA, a protein that activates the tyrosyl phosphatase activity of protein phosphatase 2A. J. Biol. Chem. 269, 15668–15675.10.1016/S0021-9258(17)40733-2Search in Google Scholar
Chao, Y., Xing, Y., Chen, Y., Xu, Y., Lin, Z., Li, Z., Jeffrey, P.D., Stock, J.B., and Shi, Y. (2006). Structure and mechanism of the phosphotyrosyl phosphatase activator. Mol. Cell 23, 535–546.10.1016/j.molcel.2006.07.027Search in Google Scholar PubMed
Chen, J., Martin, B.L., and Brautigan, D.L. (1992). Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257, 1261–1264.10.1126/science.1325671Search in Google Scholar PubMed
Chen, Y., Xu, Y., Bao, Q., Xing, Y., Li, Z., Lin, Z., Stock, J.B., Jeffrey, P.D., and Shi, Y. (2007). Structural and biochemical insights into the regulation of protein phosphatase 2A by small t antigen of SV40. Nat. Struct. Mol. Biol. 14, 527–534.10.1038/nsmb1254Search in Google Scholar PubMed
Chen, V.B., Arendall, W.B., 3rd, Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21.10.1107/S0907444909042073Search in Google Scholar PubMed PubMed Central
Cho, U.S. and Xu, W. (2007). Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445, 53–57.10.1038/nature05351Search in Google Scholar PubMed
Cho, U.S., Morrone, S., Sablina, A.A., Arroyo, J.D., Hahn, W.C., and Xu, W. (2007). Structural basis of PP2A inhibition by small t antigen. PLoS Biol. 5, e202.10.1371/journal.pbio.0050202Search in Google Scholar PubMed PubMed Central
De Baere, I., Derua, R., Janssens, V., Van Hoof, C., Waelkens, E., Merlevede, W., and Goris, J. (1999). Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue. Biochemistry 38, 16539–16547.10.1021/bi991646aSearch in Google Scholar PubMed
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol. NMR 6, 277–293.10.1007/BF00197809Search in Google Scholar PubMed
DeLano, W. (2003). The PyMOL Molecular Graphics System. DeLano Scientific LLC. http://www.pymol.org.Search in Google Scholar
Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D 66, 486–501.10.1107/S0907444910007493Search in Google Scholar PubMed PubMed Central
Fellner, T., Lackner, D.H., Hombauer, H., Piribauer, P., Mudrak, I., Zaragoza, K., Juno, C., and Ogris, E. (2003). A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo. Genes Dev. 17, 2138–2150.10.1101/gad.259903Search in Google Scholar
Gallego, M. and Virshup, D.M. (2005). Protein serine/threonine phosphatases: life, death, and sleeping. Curr. Opin. Cell Biol. 17, 197–202.10.1016/j.ceb.2005.01.002Search in Google Scholar
Graslund, S., Sagemark, J., Berglund, H., Dahlgren, L.G., Flores, A., Hammarstrom, M., Johansson, I., Kotenyova, T., Nilsson, M., Nordlund, P., et al. (2008). The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr. Purif. 58, 210–221.10.1016/j.pep.2007.11.008Search in Google Scholar
Groves, M.R., Hanlon, N., Turowski, P., Hemmings, B.A., and Barford, D. (1999). The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96, 99–110.10.1016/S0092-8674(00)80963-0Search in Google Scholar
Grzesiek, S., Stahl, S.J., Wingfield, P.T., and Bax, A. (1996). The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR. Biochemistry 35, 10256–10261.10.1021/bi9611164Search in Google Scholar
Guo, F., Stanevich, V., Wlodarchak, N., Sengupta, R., Jiang, L., Satyshur, K.A., and Xing, Y. (2014). Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone. Cell Res. 24, 190–203.10.1038/cr.2013.138Search in Google Scholar
Heijman, J., Dewenter, M., El-Armouche, A., and Dobrev, D. (2013). Function and regulation of serine/threonine phosphatases in the healthy and diseased heart. J. Mol. Cell Cardiol. 64, 90–98.10.1016/j.yjmcc.2013.09.006Search in Google Scholar
Huhn, J., Jeffrey, P.D., Larsen, K., Rundberget, T., Rise, F., Cox, N.R., Arcus, V., Shi, Y., and Miles, C.O. (2009). A structural basis for the reduced toxicity of dinophysistoxin-2. Chem. Res. Toxicol. 22, 1782–1786.10.1021/tx9001622Search in Google Scholar
Hunter, T. (1995). Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225–236.10.1016/0092-8674(95)90405-0Search in Google Scholar
Ikehara, T., Ikehara, S., Imamura, S., Shinjo, F., and Yasumoto, T. (2007). Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B). Biochem. Biophys. Res. Commun. 354, 1052–1057.10.1016/j.bbrc.2007.01.085Search in Google Scholar PubMed
Janssens, V. and Goris, J. (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439.10.1042/bj3530417Search in Google Scholar
Janssens, V., Longin, S., and Goris, J. (2008). PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends Biochem. Sci. 33, 113–121.10.1016/j.tibs.2007.12.004Search in Google Scholar PubMed
Jiang, L., Stanevich, V., Satyshur, K.A., Kong, M., Watkins, G.R., Wadzinski, B.E., Sengupta, R., and Xing, Y. (2013). Structural basis of protein phosphatase 2A stable latency. Nat. Commun. 4, 1699.10.1038/ncomms2663Search in Google Scholar PubMed PubMed Central
Johnson, B.A. and Blevins, R.A. (1994). NMR View: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614.10.1007/BF00404272Search in Google Scholar PubMed
Kabsch, W. (2010). XDS. Acta Crystallogr. D 66, 125–132.10.1107/S0907444909047337Search in Google Scholar PubMed PubMed Central
Lambrecht, C., Haesen, D., Sents, W., Ivanova, E., and Janssens, V. (2013). Structure, regulation, and pharmacological modulation of PP2A phosphatases. Methods Mol. Biol. 1053, 283–305.10.1007/978-1-62703-562-0_17Search in Google Scholar PubMed
Leulliot, N., Vicentini, G., Jordens, J., Quevillon-Cheruel, S., Schiltz, M., Barford, D., van Tilbeurgh, H., and Goris, J. (2006). Crystal structure of the PP2A phosphatase activator: implications for its PP2A-specific PPIase activity. Mol. Cell 23, 413–424.10.1016/j.molcel.2006.07.008Search in Google Scholar PubMed
Löw, C., Jegerschold, C., Kovermann, M., Moberg, P., and Nordlund, P. (2012). Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1. PLoS One 7, e38244.Search in Google Scholar
Magnusdottir, A., Stenmark, P., Flodin, S., Nyman, T., Kotenyova, T., Graslund, S., Ogg, D., and Nordlund, P. (2009). The structure of the PP2A regulatory subunit B56γ: the remaining piece of the PP2A jigsaw puzzle. Proteins 74, 212–221.10.1002/prot.22150Search in Google Scholar PubMed
McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674.10.1107/S0021889807021206Search in Google Scholar PubMed PubMed Central
Mumby, M.C. and Walter, G. (1993). Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol. Rev. 73, 673–699.10.1152/physrev.1993.73.4.673Search in Google Scholar PubMed
Ogris, E., Du, X., Nelson, K.C., Mak, E.K., Yu, X.X., Lane, W.S., and Pallas, D.C. (1999). A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A. J. Biol. Chem. 274, 14382–14391.10.1074/jbc.274.20.14382Search in Google Scholar PubMed PubMed Central
Quistgaard, E.M., Nordlund, P., and Löw, C. (2012). High-resolution insights into binding of unfolded polypeptides by the PPIase chaperone SlpA. FASEB J. 26, 4003–4013.10.1096/fj.12-208397Search in Google Scholar PubMed
Rempola, B., Kaniak, A., Migdalski, A., Rytka, J., Slonimski, P.P., and di Rago, J.P. (2000). Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae. Mol. Gen. Genet. 262, 1081–1092.10.1007/PL00008651Search in Google Scholar
Sablina, A.A., Hector, M., Colpaert, N., and Hahn, W.C. (2010). Identification of PP2A complexes and pathways involved in cell transformation. Cancer Res. 70, 10474–10484.10.1158/0008-5472.CAN-10-2855Search in Google Scholar PubMed PubMed Central
Schmitz, M.H., Held, M., Janssens, V., Hutchins, J.R., Hudecz, O., Ivanova, E., Goris, J., Trinkle-Mulcahy, L., Lamond, A.I., Poser, I., et al. (2010). Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-β1 as key mitotic exit regulators in human cells. Nat. Cell Biol. 12, 886–893.10.1038/ncb2092Search in Google Scholar PubMed PubMed Central
Sents, W., Ivanova, E., Lambrecht, C., Haesen, D., and Janssens, V. (2013). The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity. FEBS J. 280, 644–661.10.1111/j.1742-4658.2012.08579.xSearch in Google Scholar PubMed
Shi, Y. (2009a). Assembly and structure of protein phosphatase 2A. Sci. China C Life Sci. 52, 135–146.10.1007/s11427-009-0018-3Search in Google Scholar PubMed
Shi, Y. (2009b). Serine/threonine phosphatases: mechanism through structure. Cell 139, 468–484.10.1016/j.cell.2009.10.006Search in Google Scholar PubMed
Stanevich, V., Jiang, L., Satyshur, K.A., Li, Y., Jeffrey, P.D., Li, Z., Menden, P., Semmelhack, M.F., and Xing, Y. (2011). The structural basis for tight control of PP2A methylation and function by LCMT-1. Mol. Cell 41, 331–342.10.1016/j.molcel.2010.12.030Search in Google Scholar PubMed PubMed Central
Tsai, M.L., Cronin, N., and Djordjevic, S. (2011). The structure of human leucine carboxyl methyltransferase 1 that regulates protein phosphatase PP2A. Acta Crystallogr. D 67, 14–24.10.1107/S0907444910042204Search in Google Scholar PubMed
Virshup, D.M. and Shenolikar, S. (2009). From promiscuity to precision: protein phosphatases get a makeover. Mol. Cell 33, 537–545.10.1016/j.molcel.2009.02.015Search in Google Scholar PubMed
Weigelt, J. (1998). Single scan, sensitivity- and gradient-enhanced TROSY for multidimensional NMR experiments (vol 120, pg 10778, 1998). J. Am. Chem. Soc. 120, 12706–12706.10.1021/ja9855287Search in Google Scholar
Wlodarchak, N., Guo, F., Satyshur, K.A., Jiang, L., Jeffrey, P.D., Sun, T., Stanevich, V., Mumby, M.C., and Xing, Y. (2013). Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation. Cell Res. 23, 931–946.10.1038/cr.2013.77Search in Google Scholar PubMed PubMed Central
Woestenenk, E.A., Hammarstrom, M., van den Berg, S., Hard, T., and Berglund, H. (2004). His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. J. Struct. Funct. Genomics 5, 217–229.10.1023/B:jsfg.0000031965.37625.0eSearch in Google Scholar
Wurzenberger, C. and Gerlich, D.W. (2011). Phosphatases: providing safe passage through mitotic exit. Nat. Rev. Mol. Cell Biol. 12, 469–482.10.1038/nrm3149Search in Google Scholar PubMed
Xing, Y., Xu, Y., Chen, Y., Jeffrey, P.D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J.B., and Shi, Y. (2006). Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, 341–353.10.1016/j.cell.2006.09.025Search in Google Scholar PubMed
Xing, Y., Li, Z., Chen, Y., Stock, J.B., Jeffrey, P.D., and Shi, Y. (2008). Structural mechanism of demethylation and inactivation of protein phosphatase 2A. Cell 133, 154–163.10.1016/j.cell.2008.02.041Search in Google Scholar PubMed
Xu, Y., Xing, Y., Chen, Y., Chao, Y., Lin, Z., Fan, E., Yu, J.W., Strack, S., Jeffrey, P.D., and Shi, Y. (2006). Structure of the protein phosphatase 2A holoenzyme. Cell 127, 1239–1251.10.1016/j.cell.2006.11.033Search in Google Scholar PubMed
Xu, Y., Chen, Y., Zhang, P., Jeffrey, P.D., and Shi, Y. (2008). Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation. Mol. Cell 31, 873–885.10.1016/j.molcel.2008.08.006Search in Google Scholar PubMed PubMed Central
Supplemental Material: The online version of this article (DOI 10.1515/hsz-2014-0106) offers supplementary material, available to authorized users.
©2014 by Walter de Gruyter Berlin/Boston
