Analyses of the Regulatory Mechanism of Porcine WEE1B: The Phosphorylation Sites of Porcine WEE1B and Mouse WEE1B Are Different

Abstract

Abstract. WEE1B, an oocyte-specific kinase, phosphorylates the CDC2 inhibitory site and maintains the meiotic arrest of oocytes at the first meiotic prophase in several mammalian species. However, the molecular mechanisms controlling WEE1B activity have not been fully examined in species other than mice. In the present study, we analyzed the regulation mechanisms of porcine WEE1B (pWEE1B), focusing on the cAMP-dependent protein kinase (PKA) phosphorylation site and intracellular localization. As the PKA phosphorylation site in mouse WEE1B (mWEE1B) was not conserved in pWEE1B, we predicted that four serine residues would be phosphorylatable by PKA in pWEE1B (Ser77, Ser118, Ser133 and Ser149) and constructed FLAG-tagged replaced-pWEE1Bs, in which each of the PKAphosphorylatable serines was mutated into a non-phosphorylatable alanine. We injected one of their mRNAs into porcine immature oocytes and found that the Ser77-replaced pWEE1B lost the WEE1B function, whereas the wild-type and other replaced-pWEE1Bs could maintain the meiotic arrest of oocytes. Next, the localization of pWEE1B was examined by immunohistochemistry, and exclusive nuclear localization was revealed in the fully grown oocytes. We generated a nuclear localization signal (NLS)-deleted pWEE1B (ΔNLS-pWEE1B) and then overexpressed it in porcine immature oocytes. We found that ΔNLS-pWEE1B was distributed uniformly in the cytoplasm and could not maintain the meiotic arrest of porcine oocytes. These results suggest that pWEE1B is activated after phosphorylation of the Ser77 residue, which is different from the phosphorylation site that activates mWEE1B; that pWEE1B is localized in the nucleus; and that the nuclear localization is essential for its function. Key words: Intracellular localization, Phosphorylation site, Porcine oocyte, Porcine WEE1B (J. Reprod. Dev. 57: [223][224][225][226][227][228] 2011) n mammals, ovarian oocytes are arrested at the first meiotic prophase until meiotic resumption is induced by luteinizing hormone stimulation. The maintenance of low activity of the maturation promoting factor (MPF) is a prerequisite for the maintenance of this meiotic arrest of oocytes. Phosphorylation of inhibitory phosphorylation sites in CDC2, a catalytic subunit of MPF, plays a principle role in inhibiting the MPF activity of these oocytes, and WEE1B, also called WEE2, has been identified as an oocyte-specific kinase involved in this phosphorylation in mice, humans, rhesus monkeys and pigs [1][2][3][4]. The induction of germinal vesicle break down (GVBD) by WEE1B inhibition using RNA interference or antisense oligonucleotide injection has been shown in mouse, rhesus monkey and porcine immature oocytes, in which spontaneous meiotic resumption was pharmacologically prevented [2][3][4][5]. Successful meiotic resumption was also induced in porcine growing oocytes, which could not start meiosis autonomously, by the injection of WEE1B antisense RNA [6]. These studies suggest that the function of WEE1B is required for maintenance of the meiotic arrest of...

Analyses of the Involvement of PKA Regulation Mechanism in Meiotic Incompetence of Porcine Growing Oocytes1

“…These results are similar to those found in the previous report where WEE1B suppression was used instead of PKA inhibition and GVBD rates were examined in CDC2-overexpressed and/or WEE1B-suppressed GOs [11]. Because PKA phosphorylates and activates WEE1B [2][3][4][5], the effect of continuous PKA activation in GOs could be mediated by continuous WEE1B activation and inhibition of CDC2 kinase by activated WEE1B. These facts indicate that CDC2 activity is inhibited both quantitatively and qualitatively rather than by either of mechanism alone, implying that a double-pronged mechanism is used by mammalian GOs in preventing an unexpected activation of CDC2.…”

“…The resumption of meiosis requires elevation of CDC2 kinase activity, although this elevation is inhibited in ovarian follicles by a high intraoocyte cAMP level and resulting high cAMPdependent protein-kinase (PKA) activity [1]. In meioticarrested mouse and pig oocytes, it has been reported that active PKA phosphorylates and activates WEE1B, an oocytespecific member of the WEE1 kinase family, and that the active WEE1B phosphorylates inhibitory phosphorylation sites of CDC2 in order to maintain its low activity [2][3][4]. The cAMP level and PKA activity in full-grown oocytes (FGOs) is decreased drastically by hormonal stimulation or isolation from follicles, resulting in inactivation of WEE1B and subsequent CDC2 activation, which induces meiotic resumption [5].…”

Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos

“…PKA is important in regulating the cell cycle progression of eukaryotic organisms. A study by Shimaoka et al suggested that continuous high PKA activity is a primary cause of the meiotic incompetence of pig-growing oocytes, and that this PKA activity is, not only caused by an insufficient expression level of PKA subunits, but may be attributed to more complex spatial-temporal regulation mechanisms (45). Our studies have demonstrated that PKA negatively regulates cell cycle progression in one-cell stage mouse embryos by inhibiting MPF (46) and CDC25B acts as a direct substrate of PKA (27).…”

Contributions of UBE2C and UBE2S to meiotic progression of porcine oocytes

“…On the other hand, although UBE2 overexpression showed a decrease in the CCNB level, the oocytes were able to reach MI, similar to the controls. In the initiation of porcine oocyte maturation, the activation of MPF is attributed to the dephosphorylation of pre-MPF, and the initiation of germinal vesicle breakdown (GVBD) can take place even with low MPF activity [ 23 , 32 ]. It was also reported in mice that inhibition or overexpression of UBE2 did not have an impact on the meiotic resumption of oocytes [ 19 ].…”