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Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes

Published online by Cambridge University Press:  01 August 2007

C. Schuon ,
S. Ebeling  and
B. Meinecke
C. Schuon
Affiliation:
Department of Reproductive Biology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
S. Ebeling
Affiliation:
Department of Reproductive Biology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
B. Meinecke*
Affiliation:
Department of Reproductive Biology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
*
All correspondence to: Burkhard Meinecke, Department of Reproductive Biology, University of Veterinary Medicine Hannover, Foundation, Buenteweg 2, D-30559 Hannover, Germany. Tel: +49 511 953 7181. Fax: + 49 511 953 7150. e-mail: [email protected]
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Summary

The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus–oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600–800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22–24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.

Keywords

MAPKMaturationp90rskPhosphorylationPig oocyte
Type
Research Article
Information
Zygote , Volume 15 , Issue 3 , August 2007 , pp. 215 - 223
Copyright
Copyright © Cambridge University Press 2007

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References

Ballou, L.M., Luther, H. & Thomas, G. (1991). MAP2 kinase and 70 K S6 kinase lie on distinct signalling pathways. Nature 349, 348–50.Google Scholar
Bannerjee, P., Ahmad, M.F., Grove, J.R., Kozlosky, C., Price, D.J. & Avruch, J. (1990). Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase. PNAS 87, 8550–4.Google Scholar
Bhatt, R.R. & Ferrell Jr, J.E. (1999). The protein kinase p90rsk as an essential mediator of cytostatic factor activity. Science 286, 1362–65.Google Scholar
Chung, J., Kuo, C.J., Crabtree, G.R. & Blenis, J. (1992). Rapamicin-FKBP specifically blocks growth-dependent activation of an signalling by the 70 kd S6 protein kinase. Cell 69, 1227–36.Google Scholar
Davis, R.J. (1993). The mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem. 268, 14553–6.Google Scholar
Ebeling, S., Bösebeck, C. & Meinecke, B. (2005). Mitogen-activated protein kinase in porcine cumulus cells. Reprod. Fert. Dev. 17, 291.Google Scholar
Erikson, E. & Maller, J.L. (1985). A protein kinase from Xeno-pus eggs specific for ribosomal protein S6. PNAS 82, 742–6.Google Scholar
Erikson, E. & Maller, J.L. (1986). Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6. J. Biol. Chem. 261, 350–55.Google Scholar
Erikson, E. & Maller, J.L. (1989). In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocytes maturation. J. Biol. Chem. 264, 13711–17.Google Scholar
Fan, H.Y., Tong, C., Lian, L., Li, S.W., Gao, W.X., Cheng, Y., Chen, D.Y., Schatten, H. & Sun, Q.Y. (2003). Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization. Biol. Reprod. 68, 968–977.Google Scholar
Frödin, M. & Gammeltoft, S. (1999). Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. M0.1 Cell. Endocrinol. 151, 65–77.Google Scholar
Gautier, J., Norbury, C., Lohka, M., Nurse, P. & Maller, J. (1988). Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell 54, 433–39.Google Scholar
Gavin, A.C. & Schorderet-Slatkine, S. (1997). Ribosomal S6 kinase p90rsk and mRNA cap-binding protein eIF4E phosphorylations correlate with MAP kinase activation during meiotic reinitiation of mouse oocytes. Mol. Reprod. Dev. 446, 383–391.3.0.CO;2-#>CrossRefGoogle Scholar
Gross, S.D., Schwab, M.S., Lewellyn, A.L. & Maller, J.L. (1999). Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90RSK. Science 286, 1365–67.CrossRefGoogle ScholarPubMed
Gross, S.D., Schwab, M.S., Taieb, F.E., Lewellyn, A.L., Qian, Y.W. & Maller, J.L. (2000). The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by P90RSk. Curr. Biol. 10, 430–38.Google Scholar
Grove, J.R., Price, D.J., Banerjee, A., Balasubramanyam, M.F., Ahmad, M.F. & Avruch, J. (1993). Regulation of an epitope-tagged recombinant RSK-1 S6 kinase by phorbol ester and ERK/MAP kinase. Biochemistry 32, 7727–38.Google Scholar
Hsiao, K.M., Chou, S.Y., Shih, S.J. & Ferell, J.E. Jr. (1994). Evidence that inactive p42 mitogen-activated protein kinase and inactive Rsk exist as a heterodimer in vivo. PNAS 91, 5480–84.Google Scholar
Hunter, R.H.F. & Polge, C. (1966). Maturation of follicular oocytes in the pig after injection of human chorionic gonadotrophin. J. Reprod. Fert. 12, 525–31.Google Scholar
Jones, S.W., Erikson, E., Blenis, J., Maller, L.M. & Erikson, R.L. (1988). A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases. PNAS 85, 3377–81.Google Scholar
Kagii, H., Naito, K., Sugiura, K., lwamori, N., Ohashi, S., Goto, S., Yamanouchi, K. & Tojo, H. (2000). Requirement of mitogen-activated protein kinase activation for the meiotic resumption of porcine oocytes. J. Reprod. Dev. 46, 249–56.Google Scholar
Kalab, P., Kubiak, J.Z., Verlhac, M.H., Colledge, W.H. & Maro, B. (1996). Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and dependent activation. Development 122, 1957–1964.Google Scholar
Kozma, S.C., Ferarri, S., Bassand, P., Siegmann, M., Trotty, N. & Thomas, G. (1990). Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily. PNAS 87, 7365–9.Google Scholar
Lohka, M.J., Hayes, M. & Maller, J.L. (1988). Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. PNAS 85, 3009–13.Google Scholar
Lu, Q., Smith, G.D., Chen, D.Y., Yang, Z., Han, Z.M., Schatten, H. & Sun, Q.Y. (2001). Phosphorylation of mitogen-activated protein kinase is regulated by protein kinase C, cyclic 3′,5′-adenosine monophosphate, and protein phosphatase modulators during meiosis resumption in rat oocytes. Biol. Reprod. 64, 1444–50.Google Scholar
Motlik, J. & Fulka, J. (1976). Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. J. Exp. Zool. 198, 155–62.CrossRefGoogle ScholarPubMed
Philipova, R. & Whitaker, M. (2005). Active ERK1 is dimerized in vivo: biphosphodimers generate peak kinase activity and monophosphodimers maintain basal ERK1 activity. J. Cell. Sci. 118, 5767–76.Google Scholar
Ratky, J., Rath, D. & Brüssow, K.P. (2003a). In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection. Acta. Vet. Hung. 51, 95–101.Google Scholar
Ratky, J., Brüssow, K.P., Torner, H. & Egerszegi, I. (2003b). Collection of in vivo matured pig oocytes. AETE Newsletter 20, 13–16.Google Scholar
Rubinfeld, H. & Seger, R. (2004). The ERK cascade as a prototype of MAPK signalling pathways. In: MAP Kinase Signalling Protocols, pp. 1–28, (ed.) R. Seger. Totowa, New Jersey, Humana Press.Google Scholar
Schwab, M.S., Kim, S.H., Terade, N., Edfäll, C., Kozma, S.C., Thomas, G. & Maller, J.L. (1999). p70S6K controls selective mRNA translation during oocyte maturation and early embryogenesis in Xenopus laevis. Mol. Cell. Biol. 19, 2485–94.Google Scholar
Su, Y.Q., Rubinstein, S., Luria, A., Lax, Y. & Breitbart, H. (2001). Involvement of MEK-mitogen-activated protein kinase pathway in follicle-stimulating hormone-induced but not spontaneous meiotic resumption of mouse oocytes. Biol. Reprod. 65, 358–65.Google Scholar
Sugiura, K., Iwamori, N., Kagii, H., Goto, S., Naruoka, H., Yada, E., Yamanouchi, K. & Tojo, H. (2002). Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation. Zygote 10, 31–36.Google Scholar
Tan, X., Chen, D.Y., Yang, Z., Wang, Y.C., Li, M., Schatten, H. & Sun, Q.Y. (2001). Phosphorylation of p90rsk during meiotic maturation and parthenogenetic activation of rat oocytes: correlation with MAP kinases. Zygote 9, 269–76.Google Scholar
Tong, C., Fan, H.Y., Chen, D.Y., Song, X.F., Schatten, H. & Sun, Q.Y. (2003). Effects Of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtubule organization, asymmetric division and metaphase II arrest. Cell. Res. 13, 375–85.Google Scholar
Verlhac, M.H., Kubiak, J.Z., Clarke, H.J. & Maro, B. (1994). Microtubule and chromatin behaviour follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 120, 1017–25.Google Scholar
Von Manteuffel, S.R., Dennis, P.B., Pullen, N., Gingras, A.C., Sonnenberg, N. & Thomas, G. (1997). The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70S6K. Mol. Cell. Biol. 17, 5426–36.Google Scholar
Wehrend, A. & Meinecke, B. (2001). Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages. Anim. Reprod. Sci. 66, 175–84.Google Scholar
Yu, H.Q., Bou, S., Chen, D.Y. & Sun, Q.Y. (2002). Phosphorylation of MAP kinase and p90rsk and its regulation during in vitro maturation of cumulus-enclosed rabbit oocytes. Zygote 10, 311–16.Google Scholar