Tuning the activation threshold of a kinase network by nested feedback loops

Abstract

Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network's response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase-3beta (GSK-3beta) as a dampener of progesterone responsiveness. GSK-3beta engages the meiotic kinase network through a double-negative feedback loop; this specific feedback architecture raises the progesterone threshold in correspondence with the strength of double-negative signaling. We also identified a marker of nutritional status, l-leucine, which lowers the progesterone threshold, indicating that oocytes integrate additional signals into their cell-fate decisions by modulating progesterone responsiveness.

Fig. 1

GSK-3β dampens oocytes’ progesterone responsiveness. (A) Overview of signal transduction during Xenopus oocyte maturation. Prog. indicates progesterone. Kinases with previously demonstrated systems-level roles are indicated in black; direct biochemical connections are designated by black arrows. Accumulation of Mos, phosphorylation of p42MAPK (Tyr²⁰⁴), and de-phosphorylation of Cdk1 (Tyr¹⁵) are biochemical markers for M phase. The mature oocyte displays germinal vesicle breakdown (GVBD), a morphological marker for M phase. (B) The structure of 7AIPM, a GSK-3β inhibitor. (C) Progesterone dose responses conducted in the presence of 7AIPM (pink and red) or the solvent dimethylsulfoxide (DMSO, gray and black). The cell-fate decisions of individual oocytes were observed at steady state. Results from three independent, paired (+ or −7AIPM) dose responses are plotted, representing 2221 oocytes scored individually (error bars represent SE). For non-normalized dose responses see (8). (Inset) Composite progesterone dose responses from the three independent experiments shown in (C). For DMSO-treated oocytes (gray and black), EC₅₀ = 1.00 (normalized) and n_H = 2.3. For 7AIPM-treated oocytes (pink and red), EC₅₀ = 0.43 (normalized to DMSO control) and n_H =2.3. (D) Representative single-oocyte Western blots. Each lane contains lysate prepared from one oocyte at steady state.

Fig. 2

GSK-3β inactivation is an M-phase event. (A) Feed-forward versus feedback models for GSK-3β–dependent progesterone desensitization, relating the activity of GSK-3β (G) to interphase (I) and M-phase (M) signaling. Relative signal strength (i.e., the amount of downstream, promeiosis signal per unit of progesterone) is indicated by the thickness of the gray and pink arrows. The pink arrow designates the threshold-regulation step. (Right) The black curve models the input dose-response curve of the native bistable system; the red curve models the response after 7AIPM treatment. (B) Time course of phospho-regulation and Mos accumulation during maturation. Average phospho-MAPK, phospho-GSK-3β, and total Mos were quantified by Western blotting (error bars are ±SD, n = approximately 24 oocytes per time point). At 9.5 hours, GVBD was 100%. (C) Single-oocyte Western blots at intermediate progesterone. Lysates were prepared from individual oocytes at steady state, then Western blotted. Lanes were assigned by cell morphology (+ indicates GVBD was observed; –, GVBD was not observed); lane lines are included for clarity. (D) MEK inhibition before progesterone stimulus. Lysate was prepared from individual oocytes at steady state. (Left) Phospho–GSK-3β signal quantitated by closed-circuit device (CCD) camera. Dots represent signal from single oocytes; lines indicate population average. PD98059 pretreatment reduces average GSK-3β Ser⁹ phosphorylation significantly (P < 0.0001, Student's t test). (Right) Phospho-MAPK and phospho-GSK in representative single oocytes. Phospho-MAPK is a direct readout of MEK activity and M-phase entry. DMSO pretreatment: 71.2% M phase; PD98059 pretreatment: 10.9% M phase. Total n = 48 (DMSO) and 46 (PD98059). (E) Phospho–GSK-3β and phospho-MAPK in oocytes microinjected with Mos or nondegradable cyclin B1 (Δ90). Oocytes were lysed individually after GVBD; lysates were run on the same gel and Western blotted on a single blot. Microinjected oocytes were not stimulated with progesterone.

Fig. 3

M-phase GSK-3β inactivation is switchlike, MEK-dependent, and irreversible. (A and B). Phosphorylation of GSK-3β in single oocytes. Phospho–GSK-3β within each oocyte was scored by Western blot (A) then quantitated by CCD camera [(B), top, red]. Lanes indicated by “+” were treated with 1.5 μM progesterone. Dots represent the phospho–GSK-3β signal from individual oocytes; lines indicate the oocyte population's average phospho–GSK-3β signal. The single-cell quantitation of MAPK phosphorylation is included for reference [(B), bottom, blue]. (C and D) Progesterone removal. After GVBD, oocytes were washed extensively over 18 hours then lysed individually. Phospho–GSK-3β was scored by Western blot [(C), a representative oocyte, total n = 23]. In (D), PD98059 was added during the washout period. PD98059 treatment during M-phase arrest reduces average GSK-3β Ser⁹ phosphorylation significantly (P < 0.0001, Student's t test). DMSO treatment: 86.4% M phase; PD98059 treatment: 4.2% M phase.

Fig. 4

Integration of progesterone and Leu signaling. (A) Progesterone dose responses of oocytes incubated in the presence of Leu (blue) or its absence (gray and black). The cell-fate decisions of individual oocytes were observed at steady state. Results plotted represent 1290 oocytes scored individually (error bars represent SE). For control (gray and black), EC₅₀ = 1.00 (normalized) and n_H = 3.2. For Leu-treated oocytes (blue), EC₅₀ = 0.57 (normalized to control) and n_H = 3.1. (B) Representative single-cell Western blots reveal response of individual oocytes to progesterone at steady state, + or −Leu. (C) Combined treatment with Leu and 7AIPM. Oocytes were treated with DMSO (n = 91), Leu (n = 92), 7AIPM (n = 89), or Leu plus 7AIPM (n = 89) before progesterone stimulus. Cell-fate decisions were scored at steady state. Error bars represent SE. Pairwise comparisons were made to the DMSO control (black asterisks) and the +Leu+7AIPM treatment (blue asterisks), *P < 0.05; ***P < 0.0001; “N.S.D” indicates no significant difference (Student's t tests). More stringent χ² analysis reveals that all values are statistically different except 7AIPM and 7AIPM+Leu.