The C-terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 complexes and is required for inhibition of G(2)/M progression.
The C-terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 complexes and is required for inhibition of G(2)/M progression.
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DOI：
10.1242/jcs.112.19.3361
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发表时间：
1999-10
影响因子：
4
通讯作者：
N. J. Wells;Nobumoto Watanabe;Tsuyoshi Tokusumi;Wei Jiang;Mark A. Verdecia;Tony Hunter
中科院分区：
文献类型：
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作者：
N. J. Wells;Nobumoto Watanabe;Tsuyoshi Tokusumi;Wei Jiang;Mark A. Verdecia;Tony Hunter
Cdc2的激活是控制有丝分裂发生的普遍事件。在高等真核生物中，Cdc2活性在一定程度上受Thr14和Tyr15的抑制磷酸化调控，由Wee1和Myt1催化，从而防止灾难性的过早进入有丝分裂。在这项研究中，我们通过对S. pombe和人类骨肉瘤细胞系的过表达研究来定义Myt1的功能。与Wee1类似，人类Myt1的过表达在两种细胞类型中都阻止了进入有丝分裂；然而，Myt1的催化活性并不是在人类细胞中观察到的细胞周期延迟所必需的。Myt1的表达仅限于增殖细胞。此外，我们检测到Myt1蛋白丰度在进入有丝分裂之前没有明显下降，这与Myt1活性的丧失相吻合。我们将单克隆抗体MPM-2识别的有丝分裂磷酸表位定位到Myt1的c端结构域。有丝分裂肽酰脯氨酸异构酶Pin1能够以磷酸化依赖的方式与该结构域结合。在人细胞过表达研究中，截断Myt1的c端结构域阻止了其诱导G(2)/M期阻滞的能力，并在体外显著降低了其磷酸化Cdc2的能力。我们证明Myt1的c端结构域是募集Cdc2所必需的，我们推断该结构域位于细胞质中，因为它可以与Cdc2相互作用并被Cdc2磷酸化。综上所述，我们认为Myt1可以通过两种方式负调控Cdc2/cyclin B1并抑制G(2)/M的进展，这两种方式都需要c端结构域；首先，Myt1可以在细胞质中结合并隔离Cdc2/cyclin B1，阻止其进入细胞核；其次，Myt1可以在Thr14和Tyr15位点磷酸化相关的Cdc2/cyclin B1，从而抑制其催化活性。
Activation of Cdc2, is the universal event controlling the onset of mitosis. In higher eukaryotes, Cdc2 activity is in part regulated by inhibitory phosphorylation of Thr14 and Tyr15, catalyzed by Wee1 and Myt1, which prevents catastrophic premature entry into mitosis. In this study we defined the function of Myt1 by overexpression studies in both S. pombe and a human osteosarcoma cell line. Similar to Wee1, overexpression of human Myt1 prevented entry into mitosis in both cell types; however, Myt1 catalytic activity was not essential for the cell cycle delay observed with human cells. Myt1 expression was restricted to proliferating cells. Furthermore, we detected no major decline in Myt1 protein abundance prior to the entry into mitosis, which coincides with the loss of Myt1 activity. We localized mitotic phosphoepitopes, recognized by the monoclonal antibody MPM-2, to the C-terminal domain of Myt1. The mitotic peptidyl-prolyl isomerase, Pin1, was able to associate with this domain in a phosphorylation-dependent manner. Truncation of the C-terminal domain of Myt1 prevented its ability to induce G(2)/M phase arrest in overexpression studies in human cells and dramatically reduced its ability to phosphorylate Cdc2 in vitro. We demonstrate that the C-terminal domain of Myt1 was required for recruitment of Cdc2, and we infer that this domain lies in the cytoplasm because it can interact with and is phosphorylated by Cdc2. In conclusion, we propose that Myt1 can negatively regulate Cdc2/cyclin B1 and inhibit G(2)/M progression by two means, both of which require the C-terminal domain; first, Myt1 can bind and sequester Cdc2/cyclin B1 in the cytoplasm preventing entry into the nucleus, and, second, it can phosphorylate associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus inhibiting its catalytic activity.