Dupré A , Daldello EM , Nairn AC , Jessus C , Haccard O .

DA10044 NIDA NIH HHS , P01DA10044 NIDA NIH HHS , P01 DA010044 NIDA NIH HHS

	Figure 2. ARPP19 is phosphorylated at S109 by PKA in prophase-arrested oocytes and is partially dephosphorylated in response to Pg.(a) ARPP19 phosphorylation at S109 in oocytes. Prophase-arrested oocytes (Pro) were treated either with Pg or injected with PKI then collected at the indicated times. Oocyte lysates were analyzed by western blot for S109-phosphorylated ARPP19 (pS109), phosphorylated MAPK (pMAPK), phosphorylated Cdk1 substrates (pCdk Sub.) and total ARPP19.(b) S109 phosphorylation level of ARPP19 was quantified using ImageJ software. The error bars represent means ± s.e.m. Asterisks (*) indicate significant difference from prophase oocytes with P<0.05 (Student’s t-test, n=4 for Pg-treated oocytes and n=3 for PKI-injected oocytes).(c) Recombinant WT-GST-ARPP is phosphorylated by PKA in prophase oocytes and is dephosphorylated following Pg stimulation. Prophase-arrested oocytes (Pro) were injected with 77.5 ng of WT-GST-ARPP then 15 min later stimulated by Pg or injected with PKI. Oocytes were collected at the indicated times, WT-GST-ARPP was pulled down and isolated fractions were immunoblotted for phosphorylated WT-GST-ARPP19 at S109 (pS109) and total ARPP (GST-ARPP).
	Figure 3. S109 phosphorylation of ARPP19 prevents the prophase release of the oocyte in response to Pg.(a) Injection of S109D-GST-ARPP inhibits GVBD induction by Pg. GVBD time-course induced by Pg in prophase-arrrested oocytes injected with 775 ng of WT-, S67A-, S109A- or S109D-GST-ARPP.(b) S109D-GST-ARPP prevents Cdk1 activation induced by Pg. Oocytes from the experiment described in panel (a) were collected at GVBD and lysates were immunoblotted for Cdc27 phosphorylation, Mos accumulation and phosphorylated MAPK (pMAPK).
	Figure 4. The inhibitory effect of injected WT-GST-ARPP on meiotic resumption depends on the concentration of Pg.(a) GVBD time course of WT- or S109D-injected oocytes in response to increased concentrations of Pg. Prophase-arrested oocytes (Pro) were injected with 600 ng of either WT- or S109D-ARPP then stimulated with 1 μM of Pg. 16 hours later, no maturation was observed. Injected oocytes were further incubated with 10 μM of Pg in the external media and the GVBD time course was followed.(b) Increasing the concentration of progesterone promotes Cdk1 activation in WT-GST-ARPP injected oocytes. Prophase-arrested oocytes (Pro) or Pg-treated oocytes (Pg) from panel (a) were collected 16 hours after 1 μM Pg stimulation or at time of GVBD in response to 10 μM Pg. Oocytes were analyzed by immunoblotting Gwl, Cyclin B2 (CycB2) upshift, phosphorylated MAPK (pMAPK) and Cdk1 phosphorylation at Y15 (pY15-Cdk1).(c) The extent of ARPP19 dephosphorylation at S109 depends on Pg concentration. Prophase-arrested oocytes were injected with 600 ng of WT-GST-ARPP then induced to mature either with 1 μM or 10 μM of Pg. Oocytes were collected after Pg addition at the indicated times. The phosphorylation at S109 and the total amount of WT-GST-ARPP were immunoblotted using a specific phospho-S109 and a GST antibody respectively. S109 phosphorylation level of ARPP19 was further quantified using ImageJ software (n=6). The error bars represent means ± s.e.m.
	Figure 5. S109 phosphorylation of ARPP19 prevents the initial activation of Cdk1 required for meiosis resumption.(a) Injection of S109D-GST-ARPP inhibits Cdk1 activation induced either by PKI or Cyclin B. Prophase-arrested oocytes (Pro) were injected with 300 ng of WT- or S109D-GST-ARPP then induced to mature by Pg or by injecting either a non-degradable form of Cyclin B (CycB) or PKI. Oocytes were collected at GVBD and Cdk1 activation was followed by immunoblotting Gwl, Cyclin B2 (CycB2), MAPK phosphorylation (pMAPK), Cdk1 phosphorylation at Y15 (pY15-Cdk1) and phosphorylated Cdk1 substrates (pCdk Sub.).(b) Injection of S109D-GST-ARPP prevents Mos-induced Cdk1 activation but does not inhibit meiotic resumption induced by S67-thiophosphorylated WT-GST-ARPP. Prophase-arrested oocytes (Pro) were injected or not with 300 ng of S109D-GST-ARPP (S109D) then induced to mature by progesterone (Pg) or by injecting either Mos or S67-thiophosphorylated WT-GST-ARPP (WT*). Oocytes were collected at GVBD and immunoblotted for Gwl, Cyclin B2 upshift (CycB2), Cdk1 phosphorylation at Y15 (pY15-Cdk1), phosphorylated MAPK (pMAPK) and phosphorylated Cdk1 substrates (pCdk Sub.).(c) Injection of S109D-GST-ARPP does not inhibit Cdk1 activation induced by K71M-Gwl. Prophase-arrested oocytes (Pro) were injected with WT-, S67A-, S109A- or S109D-GST-ARPP and meiotic maturation was triggered by the injection of mRNA encoding K71M-Gwl. Oocytes were collected 18 hours later and immunoblotted for phosphorylated Cdc27, Mos accumulation and phosphorylated MAPK (pMAPK).
	Figure 6. The MPF autoamplification loop is independent of ARPP19 phosphorylation at S109.(a) S109D-GST-ARPP does not inhibit Cdk1 activation induced by okadaic acid injection (OA) or cytoplasmic transfer (CT). Prophase-arrested oocytes (Pro) were injected with 400 ng of WT- or S109D-GST-ARPP (S109D) then induced to mature either with Pg, by injecting OA or by transferring cytoplasm from MII-arrested oocytes (CT). oocytes treated with Pg, injected with OA or CT were previously incubated with IBMX for one hour. Oocytes were collected at time of GVBD and immunoblotted for Gwl, Cyclin B2 upshift (CycB2), Cdk1 phosphorylation at Y15 (pY15-Cdk1), phosphorylated MAPK (pMAPK) and S67-phosphorylated ARPP (pS67).(b) Prophase-arrested oocytes (Pro) were injected with 400 ng of S67A-, S109A-, S109D-, S109A/S67A- or S109D/S67A-GST-ARPP then induced to mature by transferring cytoplasm from MII-arrested oocytes (CT). Some oocytes were induced to mature with Pg as a control. Oocytes were collected at GVBD and immunoblotted for Gwl, Cdk1 phosphorylation at Y15 (pY15-Cdk1), phosphorylated MAPK (pMAPK), S67 phosphorylation of GST-ARPP (pS67) and GST (GST-ARPP).
	Figure 7. Schematic representation of the regulation of meiotic resumption by ARPP19 in Xenopus oocytes.During oogenesis, the phosphorylation of ARPP19 by PKA at S109 is responsible for arresting oocyte in prophase I. In response to Pg, PKA activity is downregulated and, as a consequence, ARPP19 is partly dephosphorylated at S109. ARPP19 dephosphorylation at S109 is necessary to generate the threshold level of active Cdk1 by either facilitating ARPP19 phosphorylation at S67 or by controlling protein synthesis. Once a starter amount of active Cdk1 is formed, it initiates the MPF autoamplification loop. Gwl is activated under the control of Cdk1 and fully phosphorylates ARPP19 at S67, launching the MPF autoamplification independently of PKA activity and, thus, of ARPP19 phosphorylation at S109. At that time, meiotic M-phase entry becomes irreversible.

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