ELIMINATION OF CDC2 PHOSPHORYLATION SITES IN THE CDC25 PHOSPHATASE BLOCKS INITIATION OF M-PHASE
ELIMINATION OF CDC2 PHOSPHORYLATION SITES IN THE CDC25 PHOSPHATASE BLOCKS INITIATION OF M-PHASE
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DOI：
10.1091/mbc.4.12.1337
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发表时间：
1993-12-01
影响因子：
3.3
通讯作者：
MALLER, JL
中科院分区：
文献类型：
--
作者：
IZUMI, T;MALLER, JL
The cdc25 phosphatase is a mitotic inducer that activates p34(cdc2) at the G2/M transition by dephosphorylation of Tyr15 in p34(cdc2). cdc25 本身也通过其磷酸化状态的周期性变化进行调节。为了阐明诱导有丝分裂的机制，使用重组蛋白研究了 cdc25 的磷酸化。在体外，cdc25 在相似的多个位点组中被细胞周期蛋白 A/p34(cdc2) 和细胞周期蛋白 B/p34(cdc2) 磷酸化。这种磷酸化会延迟其电泳迁移率，并激活其在体外将细胞周期蛋白 B/p34(cdc2) 激酶活性提高三至四倍的能力，正如在 M 相提取物中的内源性非洲爪蟾 cdc25 中所发现的那样。非洲爪蟾和人类 cdc25 之间保守的苏氨酸和丝氨酸残基以及随后的脯氨酸已发生突变。 Thr48、Thr67 和 Thr138 的三重突变以及这三个苏氨酸残基加上 Ser205 和 Ser285 的五重突变几乎完全消除了与 M 期提取物孵育或被 p34(cdc2) 磷酸化后 cdc25 电泳迁移率的变化。这些突变通过 p34 磷酸化分别抑制 70% 和 90% 的 cdc25 激活。在生理浓度下，这些突变体不能激活 cdc25 免疫去除的卵母细胞提取物中的细胞周期蛋白 B/p34(cdc2)，这表明 cdc2 和 cdc25 之间的正反馈循环对于细胞周期蛋白 B/p34(cdc2) 的完全激活是必要的，从而诱导突然进入体内有丝分裂。
The cdc25 phosphatase is a mitotic inducer that activates p34(cdc2) at the G2/M transition by dephosphorylation of Tyr15 in p34(cdc2). cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34(cdc2) and cyclin B/p34(cdc2) at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34(cdc2) kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34(cdc2). These mutations inhibit the activation of cdc25 by phosphorylation with p34 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34(cdc2) in cdc25-immunodepleted oocyte extracts, suggesting that a positive feedback loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34(cdc2) that induces abrupt entry into mitosis in vivo.