THE NIMA PROTEIN KINASE IN MITOTIC REGULATION

我们对细胞周期调控的理解正以惊人的速度加速。这些新的见解对我们对癌症的理解和治疗产生了重大影响。随着对细胞周期控制的进一步了解，这种影响可能会增加。NIMA蛋白激酶在调节中性曲霉的细胞周期中起关键作用。越来越多的数据表明，与NIMA相关的激酶也调节人类的细胞周期。这项拨款概述了旨在进一步加深我们对细胞周期控制的理解的实验。与NIMA相互作用并引起细胞周期阻滞的新高度保守蛋白将被分析。我们将分析NIMA在程序性细胞死亡中的作用，并验证NIMA通过促进DNA缩聚而在程序性细胞死亡中发挥作用的假设。有丝分裂时，组蛋白H3的磷酸化对DNA凝聚至关重要，这一假设将在毛竹中得到验证。我们还将验证NIMA是有丝分裂时磷酸化组蛋白H3的激酶的假设。如果NIMA不是H3激酶，则将分离该激酶，并研究NIMA对有丝分裂H3磷酸化的调节作用，因为有丝分裂H3磷酸化依赖于NIMA并由NIMA促进。NIMA通过相互作用或控制凝聚蛋白促进DNA凝聚的假设也有待检验。先前的工作已经证明MPF的酪氨酸磷酸化是一个高度保守的机制，以防止过早有丝分裂。然而，对有丝分裂起始的其他水平的检查点控制确实存在，因为假单胞杆菌菌株无法酪氨酸磷酸化MPF在细胞周期中正常进行，并且如果DNA复制停止，也会阻止有丝分裂起始。为了分离出额外的调控功能，我们计划进行基因筛选，以分离出新的有丝分裂调控因子，这些调控因子不通过MPF的酪氨酸磷酸化发挥作用，但可能调控NIMA。初步研究表明，在有丝分裂过程中，NIMA首先定位于DNA，然后定位于纺锤体，最后定位于纺锤体极体。实验概述了NIMA在固定细胞和活细胞中的定位，并定义了NIMA内负责其细胞周期特异性定位和降解的区域。最后，核NIMA促进MPF向核转移的能力将被测试。

Our understanding of cell cycle regulation has accelerated at an amazing pace. These new insights have had a major impact upon our understanding and treatment of cancer. This impact is likely to increase with further understanding of cell cycle control. The NIMA protein kinase plays a pivotal role in regulating the cell cycle in Aspergillus nidulans. Increasing data indicate that NIMA related kinases regulate the cell cycle in humans as well. This grant outlines experiments aimed at furthering our understanding of cell cycle control. New highly conserved proteins that interact with NIMA and cause cell cycle arrest are to be analyzed. The role of NIMA in programmed cell death is to be analyzed and the hypothesis tested that NIMA plays a role in programmed cell death by promoting DNA condensation. The hypothesis that phosphorylation of histone H3 is essential for DNA condensation at mitosis is to be tested in A. nidulans. We will also test the hypothesis that NIMA is the kinase that phosphorylates histone H3 at mitosis. If NIMA is not the H3 kinase then this kinase will be isolated and its regulation by NIMA studied as mitotic H3 phosphorylation is dependent upon NIMA and is promoted by NIMA. The hypothesis that NIMA promotes DNA condensation by interacting or controlling condensin is also to be tested. Previous work has demonstrated that tyrosine phosphorylation of MPF is a highly conserved mechanism to prevent premature mitosis. However, other levels of checkpoint control over mitotic initiation do exist because A. nidulans strains unable to tyrosine phosphorylate MPF progress normally through the cell cycle and also arrest mitotic initiation if DNA replication is stopped. To isolate additional regulatory functions we plan genetic screens that will isolate new regulators of mitosis that do not function through tyrosine phosphorylation of MPF but may regulate NIMA. Preliminary studies indicate that during mitosis NIMA localizes first to the DNA, then the spindle and finally to the spindle pole body. Experiments are outlined to define the localization of NIMA in fixed and live cells and to define domains within NIMA that are responsible for its cell cycle specific localization and degradation. Finally, the ability of nuclear NIMA to promote translocation of MPF to the nucleus will be tested.