Two novel competing pathways establish the threshold for cyclin B-Cdk1 activation at the meiotic G2/M transition

Abstract

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K–Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B–Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B–Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K–Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B–Cdk1 activation at the meiotic G2/M transition

“…A newly generated phospho-specific antibody detects A-loop phosphorylation of starfish Akt (sfAkt) and of starfish SGK (sfSGK) In previous studies (Hiraoka et al, 2011(Hiraoka et al, , 2016, we monitored phosphorylation of the HM by immunoblotting as a marker of Akt activity in starfish oocytes because of lack of an antibody that detects the phosphorylated A-loop. Nevertheless, it is important to analyze A-loop phosphorylation to precisely evaluate Akt activation.…”

“…Nevertheless, our recent observations suggest that the PI3K-Akt pathway is insufficient (Hiraoka et al, 2016). Expression of a constitutively active form of Akt induces the meiotic G2/M transition, but its required expression level is 40-fold higher than that of endogenous Akt.…”

“…Dependent on PI-345P 3 , phosphoinositide-dependent kinase 1 (PDK1) and target of rapamycin complex 2 (TORC2) phosphorylate Akt in its activation loop (A-loop) and hydrophobic motif (HM), respectively, leading to its activation (Hiraoka et al, 2004(Hiraoka et al, , 2011. Thereafter, Akt inactivates Myt1 by directly phosphorylating Ser75 (Okumura et al, 2002) and activates Cdc25 by directly phosphorylating multiple residues including Ser188 (Hiraoka et al, 2016), resulting in reversal of the balance of Cdc25 and Myt1 activities, and consequently initial activation of cyclin B-Cdk1 (Okumura et al, 1996(Okumura et al, , 2002. These observations suggest that Akt likely represents the trigger kinase at the meiotic G2/M transition in starfish oocytes (Okumura et al, 2002).…”

“…Furthermore, expression of a constitutively active form of PI3K (CA-PI3K) induces Akt activation but results in low levels of Cdc25 and Myt1 phosphorylation, and therefore fails to induce the meiotic G2/M transition. We reported that another pathway, called "the atypical pathway," which is activated by Gβγ in parallel with PI3K activation, compensates for this insufficiency (Hiraoka et al, 2016). Although the molecular details of this pathway remain unclear, we proposed that it cooperates with the PI3K-Akt pathway to trigger cyclin B-Cdk1 activation (Hiraoka et al, 2016).…”

Protein kinase Cδ (PKCδ) involved in the regulation of pAkt1 (Ser473) on the release of mouse oocytes from diplotene arrest

“…prophase I or GV stage. 30,31 During the mitosis, Akt may regulate the intracellular localization and activation of Cdc2 by phosphorylating Cdc25B. 28,29 Aberrant activation of survival-signalling pathways plays a key role in cell cycle regulation and may cause uncontrolled cell proliferation or apoptosis.…”