In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.
- cell cycle
- insulin action
- progesterone signalling
Abbreviations used: DEPC, diethyl pyrocarbonate; DTT, dithiothreitol; ERK-2/MAPK, extracellular signal-related kinase-2 mitogen-activated protein kinase; GSK, glycogen synthase kinase; GST, glutathione S-transferase; GVBD, germinal vesicle break down; HA, haemagglutinin; IGF-I, insulin-like growth factor 1; MEK, MAPK/ERK kinase; MPF, maturation promoting factor; myr, myristoylated; PDE, phosphodiesterase; PDK-1, phosphoinositide-dependent kinase-1; PH, pleckstrin homology; PI 3-kinase, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; RT-PCR, reverse transcription PCR; xAkt, Xenopus Akt.
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