The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes

Abstract

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

Figure 1

Comparison of oocyte maturation induced by progesterone or by microinjection of PKI. Oocytes were treated with progesterone (10 μg/ml; Pg) or microinjected with 40 nl of PKI (1.5 mg/ml) and incubated at 22°C. At the indicated times groups of six oocytes were frozen. Extracts were prepared and analyzed for histone H1 kinase and Plx1 activities (A) or immunoblotted for Cdc25C, Mos, and active (phosphorylated) MAPK (pMAPK) as indicated (B). The upper (shifted) form of Cdc25C has previously been demonstrated to reflect phosphorylation and activation of the enzyme (Izumi et al., 1992; Kumagai and Dunphy, 1992).

Figure 2

Induction of the G₂/M transition in prophase extracts by PKI. Prophase extracts were supplemented with PKI (final concentration 15 μg/ml) or buffer and incubated at ambient temperature. At the indicated times samples were taken, diluted, and frozen. Samples were assayed for histone H1 kinase and Plx1 activities (A) or immunoblotted for Cdc25C, Mos, and pMAPK as indicated (B).

Figure 3

The Mos-MAPK pathway is not required for the G₂/M transition in response to PKI. U0126 or cycloheximide (CHX) was added to prophase extracts to a final concentration of 50 μM or 0.1 mg/ml, respectively. DMSO (0.5 μl–50 μl of extract) served as a control (control). Then, PKI was added, samples were frozen at the indicated times and assayed for histone H1 kinase and Plx1 activities (A) or immunoblotted for Cdc25C, Mos, and pMAPK as indicated (B).

Figure 4

The Mos-MAPK pathway is not sufficient for induction of the G₂/M transition. Prophase extracts were supplemented with either active GST-Mos (50 μg/ml final concentration) or with PKI, and samples were frozen at the indicated times, analyzed for Plx1 and histone H1 kinase activity (A), immunoblotted for Cdc25C and pMAPK (B), and for MAPK (C).

Figure 5

Plx1 is required for activation of Cdc25C and the G₂/M transition. Prophase extracts were treated with either control IgG- or anti-Plx1-coupled beads as described in MATERIALS AND METHODS. The extract supernatants were then supplemented with either PKI or PKI plus recombinant Plx1 (25 μg/ml final concentration), and samples were frozen at the indicated times and analyzed. (A) Samples were immunoblotted for Plx1 before immunodepletion (Start) and after treatment with control IgG- or anti-Plx1–coupled beads (IgG and antibody [Ab], respectively) at the indicated times. Samples were also assayed for histone H1 kinase activity (B) or immunoblotted for Cdc25C, Mos, and pMAPK (C). A sample of the Plx1 preparation was subjected to SDS-PAGE and Coomassie blue staining to assess its purity (B, right).

Figure 6

Cyclin B-Cdc2 activity is not required for Plx1 activity or the phosphorylation of Cdc25C in M phase. Prophase extracts were supplemented with PKI and incubated for 6 h. Then, samples of the extracts were either untreated (control) or further supplemented with either buffer or GST-p21^Cip1 (80 μg/ml final concentration; p21). Samples were taken at the indicated times for assay of Plx1 activity and histone H1 kinase activity (A) or immunoblotted for Cdc25C, Mos, and active MAPK (B). The arrows in A depict the time of addition of GST-p21^Cip1.