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,James L Maller

doi:10.1074/jbc.m412442200

Patrick A Eyers, Junjun Liu + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m412442200

Copy DOI

Abstract

Vertebrate oocytes are arrested in G(2) phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G(2) block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G(2) 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 G(2) 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 G(2)/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 G(2) remains an important goal for future work.

Highlights

The oocytes of most species undergo a prolonged arrest in the cell cycle during oocyte growth
In vitro kinase assays confirmed that a kinase-dead variant of PKAc in which the lysine in kinase subdomain II that interacts with the ␤-phosphates of ATP was mutated to histidine (K72H) had no detectable activity when compared with WT PKAc, whereas a PKAc mutant impaired for interaction with the RII regulatory subunit and PKI (R133A) [23, 24] or the RI regulatory subunit (D328A) [25], had enzymatic activity equivalent to that of the wild type enzyme (Fig. 1A)
The results presented in this paper are of interest in relation to the hypothesis that the inhibition of oocyte maturation and maintenance of G2 arrest by PKAc occurs by a kinaseindependent mechanism [17]

Summary

Introduction

The oocytes of most species undergo a prolonged arrest in the cell cycle during oocyte growth. Inhibition of Oocyte Maturation by PKA block GVBD induced by microinjection of either recombinant Mos protein, a MEKK that activates the MAPK pathway, or of Cdc25C, the phosphatase that directly dephosphorylates and activates Cdc2 [17, 18]. Both the Mos/MAPK pathway and the Cdc25C activation pathway are critical regulators of GVBD in Xenopus (for review see Ref. 19). The data are not consistent with the hypothesis that PKAc inhibits GVBD by a kinaseindependent mechanism

Methods

Results

Conclusion