Labbé J C, Picard A, Karsenti E, Dorée M
CNRS, LP 8402 and INSERM U.249, Montpellier, France.
Dev Biol. 1988 May;127(1):157-69. doi: 10.1016/0012-1606(88)90197-2.
The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of AMP-PNP (adenylyl-imidodiphosphate), prevents spontaneous kinase activation in gel-filtered extracts. We propose that MPF activates the M-phase-specific protein kinase in the intact cell by inactivating a factor which requires phosphorylation conditions to inhibit the prokinase to kinase transition.
一种不依赖钙离子和环核苷酸的蛋白激酶（可催化一组内源性蛋白的过度磷酸化，其中包括一种95 kDa的可溶性磷蛋白）的活性，在非洲爪蟾卵母细胞和激活卵的减数分裂和有丝分裂细胞周期中均有波动。该活性在M期较高，在间期几乎检测不到。在四个纯化步骤（硫酸铵沉淀、DEAE-纤维素层析、TSK G3000高效液相色谱和CM-琼脂糖层析）中，该活性与一种主要的组蛋白激酶共同纯化。这表明单一酶对内源性蛋白和添加的组蛋白均具有活性。在减数分裂成熟和早期胚胎细胞周期中，通过显微注射用32P-磷酸盐脉冲标记的卵母细胞中，体外检测到的M期特异性蛋白激酶活性变化与蛋白磷酸化程度变化相关。这表明该激酶具有广泛的特异性，并在向M期转变时发生的蛋白磷酸化增加中起关键作用。将成熟促进因子（MPF）显微注射到未成熟卵母细胞中，在10分钟的延迟期后，即使在没有蛋白质合成的情况下，也会触发M期特异性激酶的激活。相反，MPF显微注射不会在第一个减数分裂细胞周期完成后被环己酰亚胺阻滞的卵母细胞中，或在通过环己酰亚胺孵育阻滞在S期的激活卵中诱导激酶激活。这表明未成熟卵母细胞含有一种无活性的激酶前体（前激酶），在随后的每个细胞周期中都会合成。在不添加MPF的情况下，如果通过凝胶过滤去除低分子量代谢物，从前中期阻滞的卵母细胞制备的高速上清液中，前激酶向激酶的转变会在2小时的延迟期后“自发”发生。添加ATP，但不添加AMP-PNP（腺苷酰亚胺二磷酸），可防止凝胶过滤提取物中的自发激酶激活。我们提出，MPF通过使一种需要磷酸化条件来抑制前激酶向激酶转变的因子失活，从而在完整细胞中激活M期特异性蛋白激酶。
