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Inhibitor-2 induced M-phase arrest in Xenopus cycling egg extracts is dependent on MAPK activation

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Abstract

The evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle.

Abbreviations

CC:

chromosome condensation

CSF:

cytostatic factor

DIC:

differential interference contrast

DMSO:

dimethyl sulfoxide

EGS:

ethylene glycolbis(succinic acid N-hydrosysuccinimide ester)

Inh-2:

inhibitor-2

MAPK:

mitogenactivated protein kinase

MEK:

MAPK/extracellular signal-regulated kinase kinase

NEBD:

nuclear envelope breakdown

PKA:

protein kinase A

p90Rsk-1:

p90 ribosomal S6 kinase-1

SAC:

spindle assembly checkpoint

References

  1. 1.

    Masui, Y. and Markert, C.L. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177 (1971) 129–145.

  2. 2.

    Ferrell, J.E., Jr. Building a cellular switch: More lessons from a good egg. Bioessays 21 (1999) 866–870.

  3. 3.

    Minshull, J., Sun, H., Tonks, N.K. and Murray, A.W. A MAP kinasedependent spindle assembly checkpoint in Xenopus egg extracts. Cell 79 (1994) 475–486.

  4. 4.

    Walter, S.A., Guadagno, T.M. and Ferrell, J.E., Jr. Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase. Mol. Biol. Cell 8 (1997) 2157–2169.

  5. 5.

    Bitangcol, J.C., Chau, A.S., Stadnick, E., Lohka, M.J., Dicken, B. and Shibuya, E.K. Activation of the p42 mitogen-activated protein kinase pathway inhibits cdc2 activation and entry into M-phase in cycling Xenopus egg extracts. Mol. Biol. Cell 9 (1998) 451–467.

  6. 6.

    Chau, A.S. and Shibuya, E.K. Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction. Biol. Cell. 90 (1998) 565–572.

  7. 7.

    Chau, A.S. and Shibuya, E.K. Inactivation of p42 mitogen-activated protein kinase is required for exit from M-phase after cyclin destruction. J. Biol. Chem. 274 (1999) 32085–32090.

  8. 8.

    Bhatt, R.R. and Ferrell, J.E., Jr. The protein kinase p90 rsk as an essential mediator of cytostatic factor activity. Science 286 (1999) 1362–1365.

  9. 9.

    Gross, S.D., Schwab, M.S., Lewellyn, A.L. and Maller, J.L. Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk. Science 286 (1999) 1365–1367.

  10. 10.

    Cohen, P.T. Protein phosphatase 1—targeted in many directions. J. Cell Sci. 115 (2002) 241–256.

  11. 11.

    Antoniw, J.F. and Cohen, P. Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle. Eur. J. Biochem. 68 (1976) 45–54.

  12. 12.

    Doonan, J.H. and Morris, N.R. The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell 57 (1989) 987–996.

  13. 13.

    Ohkura, H., Adachi, Y., Kinoshita, N., Niwa, O., Toda, T. and Yanagida, M. Cold-sensitive and caffeine-supersensitive mutants of the Schizosaccharomyces pombe dis genes implicated in sister chromatid separation during mitosis. EMBO J. 7 (1988) 1465–1473.

  14. 14.

    Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T. and Yanagida, M. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell 57 (1989) 997–1007.

  15. 15.

    Ishii, K., Kumada, K., Toda, T. and Yanagida, M. Requirement for PP1 phosphatase and 20S cyclosome/apc for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+. EMBO J. 15 (1996) 6629–6640.

  16. 16.

    Hisamoto, N., Sugimoto, K. and Matsumoto, K. The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M. Mol. Cell. Biol. 14 (1994) 3158–3165.

  17. 17.

    Vanoosthuyse, V. and Hardwick, K.G. A novel protein phosphatase 1-dependent spindle checkpoint silencing mechanism. Curr. Biol. 19 (2009) 1176–1181.

  18. 18.

    Pinsky, B.A., Nelson, C.R. and Biggins, S. Protein phosphatase 1 regulates exit from the spindle checkpoint in budding yeast. Curr. Biol. 19 (2009) 1182–1187.

  19. 19.

    Fernandez, A., Brautigan, D.L. and Lamb, N.J. Protein phosphatase type 1 in mammalian cell mitosis: Chromosomal localization and involvement in mitotic exit. J. Cell Biol. 116 (1992) 1421–1430.

  20. 20.

    Wu, J.Q., Guo, J.Y., Tang, W., Yang, C.S., Freel, C.D., Chen, C., Nairn, A.C. and Kornbluth, S. PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation. Nat. Cell Biol. 11 (2009) 644–651.

  21. 21.

    Eto, M., Leach, C., Tountas, N.A. and Brautigan, D.L. Phosphoprotein inhibitors of protein phosphatase-1. Methods Enzymol. 366 (2003) 243–260.

  22. 22.

    Huang, F.L. and Glinsmann, W.H. Separation and characterization of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle. Eur. J. Biochem. 70 (1976) 419–426.

  23. 23.

    Hemmings, B.A., Resink, T.J. and Cohen, P. Reconstitution of a Mg-ATPdependent protein phosphatase and its activation through a phosphorylation mechanism. FEBS Lett. 150 (1982) 319–324.

  24. 24.

    Resink, T.J., Hemmings, B.A., Tung, H.Y. and Cohen, P. Characterisation of a reconstituted Mg-ATP-dependent protein phosphatase. Eur. J. Biochem. 133 (1983) 455–461.

  25. 25.

    DePaoli-Roach, A.A. Synergistic phosphorylation and activation of ATPMg-dependent phosphoprotein phosphatase by F A/GSK-3 and casein kinase II (PC0.7). J. Biol. Chem. 259 (1984) 12144–12152.

  26. 26.

    Wang, Q.M., Guan, K.L., Roach, P.J. and DePaoli-Roach, A.A. Phosphorylation and activation of the ATP-Mg-dependent protein phosphatase by the mitogen-activated protein kinase. J. Biol. Chem. 270 (1995) 18352–18358.

  27. 27.

    Ballou, L.M., Brautigan, D.L. and Fischer, E.H. Subunit structure and activation of inactive phosphorylase phosphatase. Biochemistry 22 (1983) 3393–3399.

  28. 28.

    Aitken, A., Hemmings, B.A. and Hofmann, F. Identification of the residues on cyclic GMP-dependent protein kinase that are autophosphorylated in the presence of cyclic AMP and cyclic GMP. Biochim. Biophys. Acta 790 (1984) 219–225.

  29. 29.

    Li, M., Stefansson, B., Wang, W., Schaefer, E.M. and Brautigan, D.L. Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle. Cell Signal. 18 (2006) 1318–1326.

  30. 30.

    Leach, C., Shenolikar, S. and Brautigan, D.L. Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis. J. Biol. Chem. 278 (2003) 26015–26020.

  31. 31.

    Brautigan, D.L., Sunwoo, J., Labbe, J.C., Fernandez, A. and Lamb, N.J. Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction. Nature 344 (1990) 74–78.

  32. 32.

    Walker, D.H., DePaoli-Roach, A.A. and Maller, J.L. Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle. Mol. Biol. Cell 3 (1992) 687–698.

  33. 33.

    Satinover, D.L., Brautigan, D.L. and Stukenberg, P.T. Aurora-A kinase and inhibitor-2 regulate the cyclin threshold for mitotic entry in Xenopus early embryonic cell cycles. Cell Cycle 5 (2006) 2268–2274.

  34. 34.

    Wang, W., Stukenberg, P.T. and Brautigan, D.L. Phosphatase inhibitor-2 balances protein phosphatase 1 and aurora B kinase for chromosome segregation and cytokinesis in human retinal epithelial cells. Mol. Biol. Cell. 19 (2008) 4852–4862.

  35. 35.

    Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 227 (1970) 680–685.

  36. 36.

    Druker, B.J., Mamon, H.J. and Roberts, T.M. Oncogenes, growth factors, and signal transduction. N. Engl. J. Med. 321 (1989) 1383–1391.

  37. 37.

    van der Velden, H.M. and Lohka, M.J. Mitotic arrest caused by the amino terminus of Xenopus cyclin B2. Mol. Cell Biol. 13 (1993) 1480–1488.

  38. 38.

    Erikson, E. and Maller, J.L. A protein kinase from Xenopus eggs specific for ribosomal protein S6. Proc. Natl. Acad. Sci. USA 82 (1985) 742–746.

  39. 39.

    Lohka, M.J., Hayes, M.K. and Maller, J.L. Purification of maturationpromoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. USA 85 (1988) 3009–3013.

  40. 40.

    Guadagno, T.M. and Ferrell, J.E., Jr. Requirement for MAPK activation for normal mitotic progression in Xenopus egg extracts. Science 282 (1998) 1312–1315.

  41. 41.

    Takenaka, K., Gotoh, Y. and Nishida, E. Map kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts. J. Cell Biol. 136 (1997) 1091–1097.

  42. 42.

    Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T. and Saltiel, A.R. PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270 (1995) 27489–27494.

  43. 43.

    Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. and Saltiel, A.R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92 (1995) 7686–7689.

  44. 44.

    Bottorff, D., Stang, S., Agellon, S. and Stone, J.C. Ras signalling is abnormal in a c-raf1 MEK1 double mutant. Mol. Cell Biol. 15 (1995) 5113–5122.

  45. 45.

    Leighton, I.A., Dalby, K.N., Caudwell, F.B., Cohen, P.T. and Cohen, P. Comparison of the specificities of p70 S6 kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: The N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation. FEBS Lett. 375 (1995) 289–293.

  46. 46.

    Tournebize, R., Andersen, S.S., Verde, F., Doree, M., Karsenti, E. and Hyman, A.A. Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis. EMBO J. 16 (1997) 5537–5549.

  47. 47.

    Vorlaufer, E. and Peters, J.M. Regulation of the cyclin B degradation system by an inhibitor of mitotic proteolysis. Mol. Biol. Cell 9 (1998) 1817–1831.

  48. 48.

    Sohaskey, M.L. and Ferrell, J.E., Jr. Distinct, constitutively active MAPK phosphatases function in Xenopus oocytes: Implications for p42 MAPK regulation in vivo. Mol. Biol. Cell 10 (1999) 3729–3743.

  49. 49.

    Wu, J.Q. and Kornbluth, S. Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era. J. Cell Sci. 121 (2008) 3509–3514.

  50. 50.

    Maller, J.L., Schwab, M.S., Gross, S.D., Taieb, F.E., Roberts, B.T. and Tunquist, B.J. The mechanism of CSF arrest in vertebrate oocytes. Mol. Cell. Endocrinol. 187 (2002) 173–178.

  51. 51.

    Chun, J., Chau, A.S., Maingat, F.G., Edmonds, S.D., Ostergaard, H.L. and Shibuya, E.K. Phosphorylation of Cdc25C by pp90Rsk contributes to a G2 cell cycle arrest in Xenopus cycling egg extracts. Cell Cycle 4 (2005) 148–154.

  52. 52.

    Li, M., Stukenberg, P.T. and Brautigan, D.L. Binding of phosphatase inhibitor-2 to prolyl isomerase Pin1 modifies specificity for mitotic phosphoproteins. Biochemistry 47 (2008) 292–300.

  53. 53.

    Satinover, D.L., Leach, C.A., Stukenberg, P.T. and Brautigan, D.L. Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein. Proc. Natl. Acad. Sci. USA 101 (2004) 8625–8630.

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Correspondence to Arian Khandani.

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Key words

  • Inhibitor-2
  • PP-1 phosphatase
  • MAPK kinase
  • p90Rsk
  • M-phase arrest
  • Xenopus cycling egg extract