Cytoplasmic Anchoring of cAMP-Dependent Protein Kinase (PKA) by A-Kinase Anchor Proteins (AKAPs) Is Required for Meiotic Arrest of Porcine Full-Grown and Growing Oocytes
Cytoplasmic Anchoring of cAMP-Dependent Protein Kinase (PKA) by A-Kinase Anchor Proteins (AKAPs) Is Required for Meiotic Arrest of Porcine Full-Grown and Growing Oocytes
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DOI：
10.1095/biolreprod.113.114736
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发表时间：
2014-03-01
影响因子：
3.6
通讯作者：
Naito, Kunihiko
中科院分区：
文献类型：
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作者：
Nishimura, Takanori;Fujii, Wataru;Naito, Kunihiko
哺乳动物的卵母细胞缺乏恢复减数分裂的能力，尽管这种限制的分子机制尚未完全了解。我们以前假设猪胚胎的减数分裂不全是由于A激酶锚蛋白（AKAP）对cAMP依赖性蛋白激酶（PKA）的复杂时空调控，但发现AKAP 1不参与猪胚胎的减数分裂不全。在本研究中，我们克隆了猪AKAP 5和AKAP 7 α的cDNA，并发现抑制这些AKAP的表达诱导PKA易位到细胞核中，并促进减数分裂的恢复，而不影响PKA的总活性，而过量表达这些AKAP没有影响。由于AKAP通过与PKA的调节亚基（PKA-R）结合来调节PKA定位，因此PKA-R与AKAP的结合被AKAP结合抑制肽抑制或PKA-R表达被反义RNA抑制。我们发现，PRKAR 1A，哺乳动物PKA-R的亚型的表达抑制和结合抑制，促进猪胚胎减数分裂的恢复，而PRKAR 2A，另一个PKA-R亚型，这些抑制没有效果。相反，PRKAR 2A的表达抑制和结合抑制对猪成熟卵母细胞减数分裂恢复的影响高于PRKAR 1A。这些结果表明，细胞质锚定PKA的AKAPs所需的减数分裂阻滞的卵母细胞和PKA-R亚型的减数分裂阻滞的维护工作从PRKAR 1A到PRKAR 2A的减数分裂能力的收购过程中改变。
Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence.