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.
Akt, also known as protein kinase B, has a central role in various signaling pathways that regulate cellular processes such as metabolism, proliferation and survival. On stimulation, phosphorylation of the activation loop (Aloop) and hydrophobic motif (HM) of Akt by the kinase phosphoinositide-dependent kinase 1 (PDK1) and the mammalian target of rapamycin complex 2 (mTORC2), respectively, results in Akt activation. A well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by mTORC2 and contributes to the stability of Akt. The role of TM phosphorylation in HM and A-loop phosphorylation has not been sufficiently evaluated. Using starfish oocytes as a model system, this study provides the first evidence that TM phosphorylation has a negative role in A-loop phosphorylation. In this system, the maturation-inducing hormone, 1-methyladenine, stimulates Akt to reinitiate meiosis through activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via introduction of exogenous human Akt (hAkt) in starfish oocytes. TM and HM phosphorylation was inhibited by microinjection of an anti-starfish TOR antibody, but not by rapamycin treatment, suggesting that both phosphorylation events depend on TORC2, as reported in mammalian cells. A single or double alanine substitution at each of three phosphorylation residues revealed that TM phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop phosphatase was inhibited by okadaic acid (OA), TM phosphorylation still reduced Aloop phosphorylation, suggesting that the effect is caused at least partially through reduction of sensitivity to PDK1. Negative regulation by TM phosphorylation was also observed in constitutively active Akt and was functionally reflected in meiosis resumption. By contrast, HM phosphorylation enhanced A-loop phosphorylation and achieved full activation of Akt via a mechanism at least partially independent of TM phosphorylation. These observations provide new insight into the mechanism controlling Akt phosphorylation in the cell.
The kinase cyclin B–Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B–Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A–Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B–Cdk1 activation, cyclin A–Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B–Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gβγ-PI3K pathway and an unidentified pathway downstream of Gβγ, called the atypical Gβγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.
Hosoda et al. show that SGK is required for an increase in intracellular pH (pHi) and cyclin B–Cdk1 activation upon meiotic resumption in starfish oocytes. The pHi increase is a prerequisite for chromosome transport and spindle assembly in ovarian oocytes.
Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.