Analyses of the regulatory mechanism of porcine WEE1B: the phosphorylation sites of porcine WEE1B and mouse WEE1B are different.
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 PKA-phosphorylatable 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.
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Takuma
T
Shimaoka
Takanori
T
Nishimura
Kiyoshi
K
Kano
Kunihiko
K
Naito
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D006801:Humans; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008540:Meiosis; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013552:Swine / Q000378:metabolism*
D018797:Cell Cycle Proteins; C509498:Wee1B protein, mouse; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D016200:Maturation-Promoting Factor