PDE3A is phosphorylated by PKB/Akt and PKA. (A) Immunoprecipitated PDE3A was incubated with or without recombinant PKB/Akt or PKA in the presence of [γ-³²P]ATP. The reaction products were subjected to SDS–PAGE and phosphorylated PDE3A (upper panel; pPDE3A) was detected after treatment of PKA or PKB/Akt. The amount of expressed PDE3A was monitored by Western blot analysis using anti-HA antibodies (lower panel; PDE3A). (B) Diagram of the constructs of PKB/Akt used in this study. (C) PDE3A-transfected (PDE3A) or empty vector-transfected (Mock) cells were cotransfected with different Akt constructs including WT, kinase-dead mutant (K179M) PKB/Akt (KD-Akt), constitutively active ΔPH myristoylated PKB/Akt (myr-Akt), or myr-Akt (A2myr-Akt). Immunoprecipitates were subjected to SDS–PAGE followed by Western blot analysis using phospho-Akt substrate antibodies. Coexpression with myr-Akt caused an increase in the phosphorylation of PDE3A (pPDE3A). The expression of PDE3A and various Akt constructs was confirmed by Western blot using anti-HA antibodies (PDE3A, Akt, Myr-Akt). (D) Coexpression of PDE3A with PKB/Akt enhances PDE3A activity. A first transfection (1st Tfn) with empty vector (Mock) or PDE3A-HA (PDE3A) was followed by a second transfection (2nd Tfn) using one of PKB/Akt constructs. PDE3A activity was enhanced by WT- or myr-Akt by 1.5- to 2-fold, but not by KD-Akt and A2myr-Akt. Expression of PDE3A and Akt constructs was analyzed by Western blot using anti-HA antibodies (PDE3A, Akt, Myr-Akt). A representative experiment of the three performed is reported. ^* and ^*** represent values of P<0.05 and P<0.005, respectively, compared to control.

An amino-terminus deletion mutant of PDE3A is not activated by PKB/Akt. (A) Putative PKB/Akt phosphorylation sites in the PDE3A amino-acid sequence. Serines marked with asterisk indicate the residues that are likely to be phosphorylated by PKB/Akt. PKB/Akt phosphorylation sites 3, 4, 5, and 6 are conserved among mouse, rat, and human PDE3A sequences. (B) MA10 cells were transfected with full-length PDE3A-HA (WT-PDE3A) or amino-terminus truncated PDE3A-HA (ΔNcoI-PDE3A) (1st Tfn), and constitutively active PKB/Akt (myr-Akt) or kinase-dead PKB/Akt (KD-Akt) was transfected after 16 hours (2nd Tfn). Nineteen hours later, cilostamide-sensitive PDE activity was measured on total cell extracts. Only full length of PDE3A not ΔNcoI-PDE3A mutant is activated by myr-Akt transfection.

S290–292 serine residues in the PDE3A sequence are important for PKB/Akt-induced PDE3A activation. (A) M3, M4, M5, M6, M3–5, M3–6, M1, 2, 6, or M1–6 mutant PDE3A-HAs were generated as described in Materials and methods. The open circle indicates the site likely to be phosphorylated by PKB/Akt. The closed circle indicates the site that was mutated from serine to alanine. Amino-acid sequences of the S290–S292 region of PDE3A and mutant M3, M4, M5, and M3–5 are shown. Underlined alanine indicates the Ser/Ala mutation introduced. (B) The phosphorylation state of the various PDE3A mutants was determined using phospho-Akt substrate antibody after cotransfection with myr-Akt and immunoprecipitation. All of the immunoprecipitated mutants were phosphorylated except M1–6, which is a mutant of all of the putative sites. (C) Cilostamide-sensitive PDE activity was measured after serial transfection of Myr-Akt or empty vector (1st Tfn) and various PDE3A mutants (2nd Tfn). All single mutated PDE3As in the M3, M4, and M5 site were not activated by PKB/Akt and only the WT and M6 mutant PDE3As were activated by myr-Akt. ^** represents P<0.01 compared to mock control. The expression levels of PDE3A and myr-Akt were confirmed with HA antibodies (lower panel; PDE3A, Myr-Akt).

A two- to four-fold increase in PDE3A activity is sufficient to induce oocyte maturation. (A) Fifty nanograms of pde3a mRNA or H₂O (vehicle) was injected into Xenopus oocytes. One group of H₂O-injected oocytes was treated with 500 nM progesterone to induce oocyte maturation. Treatment of progesterone in the H₂O-injected oocytes or PDE3A expression induces the phosphorylation of ERK (pERK), which is an established oocyte maturation marker. Protein expression (PDE3A) and the amount of loaded protein (ERK) were monitored by Western blot analysis. (B) Xenopus oocytes were injected with different amounts of pde3a mRNA (0.1–50 ng/oocyte) and treated with insulin. Twelve hours later, oocyte maturation (GVBD) was scored by the appearance of a white spot on the animal pole of oocytes. By injection with 5 ng WT pde3a mRNA (WT), more than 70% oocytes undergo GVBD, whereas only 40% of GVBD occurred by injection of 10 ng of the M3–5 mutant form (M3–5). The capacity of oocyte maturation is tested in H₂O-injected oocyte by insulin or progesterone treatment (H₂O+Ins, H₂O+Prog). (C) Xenopus oocytes were injected with 10 ng of various mutants of PDE3A and treated with 1 μM of insulin. The maturation of the oocytes was scored and PDE3A activity was measured using the oocyte lysate. The WT and M6 mutant induced more than 90% of GVBD (middle panel) and this induction of maturation was associated with an approximate two- to four-fold increase of PDE activity (upper panel). Expression levels of the PDE3As were confirmed with HA antibodies (lower panel, PDE3A).

PDE3A expression in mouse oocytes. Increasing number of WT oocytes (lanes 3–7), WT (lane 8) or pde3^−/− oocytes (lane 9, 112 oocytes/lane), and different amounts of lung lysate (lanes 12–15) were subjected to SDS–PAGE and protein detected with a PDE3A antibody (PDE3ACY). Different forms of PDE3A are expressed in the mouse oocyte. The double arrows indicate the 136 and 120 kDa specific bands. These two bands were not detected in oocyte (lane 9) and lung from pde3^−/− animals (lanes 13, 15). Full-length recombinant PDE3A-transfected Hek293 cell lysate was used as size control (lanes 2, 11).

PKB/Akt and PDE3A activation precedes activation of the MPF complex. (A) Phosphorylation state of PKB/Akt at indicated hours after hCG injection was determined using Akt phosho-S473-specific antibodies (P-S473-Akt) and the amount of loaded protein was confirmed with Akt-specific antibodies (Tot Akt). Highly phosphorylated state of Akt is detected after 2 h from hCG injection. The value of p-Akt reports the ratio of phosphorylated Akt and total Akt (square). Activity of MPF complex (Cdc2/CyclinB) was measured with five randomly selected oocytes at each time point (triangle). The phosphorylation of histone H1, which is a substrate of MPF complex, started at 2 h after hCG injection and reached a maximum at 3 h (MPF). GVBD was counted by disappearance of the germinal vesicle or extrusion of the first polar body (filled circle). A representative of the three independent experiments performed is reported. ^*** represents P<0.005 compared to 0 h. (B) The mouse ovaries were removed and lysates immunoprecipitated with PDE3A antibody. The PDE3A activity was measured in the immune complex. After 2.5 h from hCG injection, 30% increase in PDE3A activity over basal was detected. The data are the mean±s.e.m. of three independent experiments. ^** represents P<0.01 compared to 0 h.

PDE3A is required for Akt-induced mouse oocyte maturation. (A) Oocytes from pde3^−/− mice were injected with mRNA of WT PDE3A, M3–5, M1,2,6, or M1–6 mutant form. Vehicle (H₂O) was injected as a control. Maturation of mouse oocytes was monitored by disappearance of the germinal vesicle or extrusion of the first polar body 19 h after mRNA injection. The injection of WT or M1,2,6 PDE3A mRNA induced meiotic resumption around 60–75% of oocytes, whereas about 30% of oocytes resumed meiosis when M3–5 or M1–6 mRNA was used. Numbers above the bars indicate the number of GVBD stage oocytes out of total injected oocytes. Protein expression of the injected mRNAs was compared with HA antibodies using 44 oocytes per each lane (left panel) and using 39 oocytes with PDE3A antibody (right panel). (B) Oocytes from WT and pde3a^−/− mice were collected in M2 media, and WT oocytes maintained in meiotic arrest with 3.5 mM hypoxanthine. Left panel: Oocytes were injected with myr-Akt mRNA or H₂O as a control. Injection of myr-Akt causes approximately 80% of meiotic resumption in WT oocytes but not in pde3^−/− mouse oocytes. Right panel: Total PDE activity was measured with injected oocytes as described in Materials and methods. Approximately, two-fold increase in PDE activity was detected in myr-Akt-injected oocytes in three independent experiments. ^*** represents P<0.005 and ^* represents P<0.05 compared to control.