Devault A, Fesquet D, Cavadore J C, Garrigues A M, Labbé J C, Lorca T, Picard A, Philippe M, Dorée M
Centre National de la Recherche Scientifique UPR 8402, Montpellier, France.
J Cell Biol. 1992 Sep;118(5):1109-20. doi: 10.1083/jcb.118.5.1109.
We have produced human cyclin A in Escherichia coli and investigated how it generates H1 kistone kinase activity when added to cyclin-free extracts prepared from parthenogenetically activated Xenopus eggs. Cyclin A was found to form a major complex with cdc2, and to bind cdk2/Eg1 only poorly. No lag phase was detected between the time when cyclin A was added and the time when H1 histone kinase activity was produced in frog extracts, even in the presence of 2 mM vanadate, which blocks cdc25 activity. Essentially identical results were obtained using extracts prepared from starfish oocytes. We conclude that formation of an active cyclin A-cdc2 kinase during early development escapes an inhibitory mechanism that delays formation of an active cyclin B-cdc2 kinase. This inhibitory mechanism involves phosphorylation of cdc2 on tyrosine 15. Okadaic acid (OA) activated cyclin B-cdc2 kinase and strongly reduced tyrosine phosphorylation of cyclin B-associated cdc2, even in the presence of vanadate. 6-dimethylamino-purine, a reported inhibitor of serine-threonine kinases, suppressed OA-dependent activation of cyclin B-cdc2 complexes. This indicates that the kinase(s) which phosphorylate(s) cdc2 on inhibitory sites can be inactivated by a phosphorylation event, itself antagonized by an OA-sensitive, most likely type 2A phosphatase. We also found that cyclin B- or cyclin A-cdc2 kinases can induce or accelerate conversion of the cyclin B-cdc2 complex from an inactive into an active kinase. Cyclin B-associated cdc2 does not undergo detectable phosphorylation on tyrosine in egg extracts containing active cyclin A-cdc2 kinase, even in the presence of vanadate. We propose that the active cyclin A-cdc2 kinase generated without a lag phase from neo-synthesized cyclin A and cdc2 may cause a rapid switch in the equilibrium of cyclin B-cdc2 complexes to the tyrosine-dephosphorylated and active form of cdc2 during early development, owing to strong inhibition of the cdc2-specific tyrosine kinase(s). This may explain why early cell cycles are so rapid in many species.
我们已在大肠杆菌中产生了人细胞周期蛋白A，并研究了将其添加到从孤雌激活的非洲爪蟾卵制备的无细胞周期蛋白提取物中时，它是如何产生组蛋白H1激酶活性的。发现细胞周期蛋白A与cdc2形成主要复合物，而与cdk2/Eg1的结合较差。在向蛙提取物中添加细胞周期蛋白A的时间与产生组蛋白H1激酶活性的时间之间未检测到延迟期，即使存在2 mM钒酸盐（它会阻断cdc25活性）也是如此。使用从海星卵母细胞制备的提取物获得了基本相同的结果。我们得出结论，在早期发育过程中活性细胞周期蛋白A-cdc2激酶的形成避开了一种抑制机制，该机制会延迟活性细胞周期蛋白B-cdc2激酶的形成。这种抑制机制涉及cdc2酪氨酸15位点的磷酸化。冈田酸（OA）激活细胞周期蛋白B-cdc2激酶，并强烈降低与细胞周期蛋白B相关的cdc2的酪氨酸磷酸化，即使在存在钒酸盐的情况下也是如此。6-二甲基氨基嘌呤是一种已报道的丝氨酸 - 苏氨酸激酶抑制剂，它抑制OA依赖的细胞周期蛋白B-cdc2复合物的激活。这表明在抑制位点磷酸化cdc2的激酶可通过磷酸化事件失活，而该磷酸化事件本身会被OA敏感的、很可能是2A型磷酸酶拮抗。我们还发现细胞周期蛋白B - 或细胞周期蛋白A - cdc2激酶可以诱导或加速细胞周期蛋白B - cdc2复合物从无活性激酶转变为活性激酶。在含有活性细胞周期蛋白A - cdc2激酶的卵提取物中，与细胞周期蛋白B相关的cdc2即使在存在钒酸盐的情况下也不会发生可检测到的酪氨酸磷酸化。我们提出，由新合成的细胞周期蛋白A和cdc2无延迟期产生的活性细胞周期蛋白A - cdc2激酶，可能会在早期发育过程中导致细胞周期蛋白B - cdc2复合物平衡迅速转变为cdc酪氨酸去磷酸化的活性形式，这是由于对cdc2特异性酪氨酸激酶的强烈抑制。这可能解释了为什么在许多物种中早期细胞周期如此迅速。
