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J. Biol. Chem., Vol. 280, Issue 26, 24339-24346, July 1, 2005

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Regulation of the G₂/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase^*

Patrick A. Eyers¶, Junjun Liu, Nobuhiro R. Hayashi||, Andrea L. Lewellyn, Jean Gautier||, and James L. Maller**

From the Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262 and ^||Department of Genetics and Development, Columbia University, New York, New York 10032

Vertebrate oocytes are arrested in G₂ phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G₂ block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G₂ arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc). It has been reported and we confirm here that microinjection of either wild type or kinase-dead K72R PKAc inhibits progesterone-dependent release of the G₂ arrest with equal potency and that inhibition can be reversed by a second injection of the heat-stable inhibitor of PKAc, PKI. However, a mutant enzyme predicted to be completely kinase-dead from the crystal structure of PKAc, K72H PKAc, was much less inhibitory when carrying additional mutations that block interaction with either type I or type II regulatory subunit. Moreover, inhibition by K72H PKAc was reversed by PKI at a 30-fold lower concentration and with more rapid kinetics compared with wild type PKAc. K72R PKAc was found to have low but detectable activity after incubation in an oocyte extract. These results indicate that inhibition of the progesterone-dependent G₂/M transition in oocytes after microinjection of dead PKAc reflects either low residual activity or binding to regulatory subunits with a resulting net increase in the level of endogenous wild type PKAc. Consistent with this hypothesis, the induction of mitosis in Xenopus egg extracts by the addition of cyclin B was blocked by wild type PKAc but not by K72H PKAc. The identification of substrates for PKAc that maintain cell cycle arrest in G₂ remains an important goal for future work.

Received for publication, November 3, 2004 , and in revised form, April 5, 2005.

^* This work was supported by National Institutes of Health Grants GM26743-24 (to J. L. M.) and GM56781 (to J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Associate of the Howard Hughes Medical Institute.

^¶ Present address: Faculty of Life Sciences, University of Manchester, The Michael Smith Bldg., Manchester M13 9PT, United Kingdom.

^** An Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. E-mail: Jim.Maller{at}uchsc.edu.

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