Characterization of JNK in Cell Cycle Control

描述（由申请人提供）：JNK（1-3）是应激活化蛋白激酶家族的成员，其通过控制细胞死亡和存活来调节细胞应激反应。我们最近的研究揭示了一个意想不到的机制，从而调控JNK的功能，我们的初步结果表明，通过其KEN盒，JNK的目标是在细胞周期依赖性的方式降解。这种靶向由Cdh 1介导，Cdh 1募集后期促进复合物或环体（APC/C）泛素连接酶以泛素化JNK，导致其蛋白酶体依赖性降解。APC/CCDh 1介导的JNK降解主要发生在退出有丝分裂和细胞周期的G1期。我们的初步数据还确定Cdc 25 C作为JNK底物在细胞周期的G2/M-G1期。JNK降解对Cdc 25 C活性和Wee 1稳定性的调节是重要的。因此，JNK（JNKKEN）的不可降解（但可以被激活）形式诱导细胞周期蛋白-B/Cdk 1活性降低，影响纺锤体和染色体动力学并延迟有丝分裂的退出。这些发现为我们的假设提供了基础，即通过Cdc 25 C的调节，JNK在控制细胞周期进程中起着重要作用，在此期间，它受到APC/CCDh 1的严格调控。我们发现JNK是一种细胞周期调节因子，这一发现提供了JNK与不受限制的细胞周期进程以及染色体不稳定性之间的未公开联系，这些联系通常在人类癌症中观察到，其中JNK作为应激激酶和细胞周期控制蛋白之间的平衡可能会改变。为了验证我们的假设，我们建议进行以下研究：目的1 -评估在细胞周期中负责JNK活性的分子决定因素，并确定JNK家族中哪些成员对细胞周期控制最重要。目的2-表征JNK对Cdc 25 C的作用，作为其控制细胞周期转换的一部分。目的3 -表征由JNK和Cdc 25 C调控的有丝分裂中的细胞学变化;目的4 -确定JNK-KEN敲入小鼠模型中JNK在细胞周期控制中的作用的生理意义。目的5 -使用遗传、生物化学和细胞生物学方法，确定JNK在应激和DNA损伤后的细胞周期检查点途径中的作用，以及在人黑素瘤中作为组成性失调JNK活性的模型。该提案汇集了密切合作的专家，以解决关键激酶JNK的重要和新颖功能。这一共同努力将为理解JNK在细胞周期进程中的作用及其对遗传毒性应激反应以及人类癌症发展的失调控制的影响提供基础。公共卫生相关性：该应用程序将测试JNK在细胞周期控制中的重要作用的假设，更重要的是，在响应DNA损伤。支持这一假设的研究来自于我们发现JNK在细胞周期的G2 M-G1期被靶向降解。JNK通过Cdc 25 C的磷酸化介导其功能，Cdc 25 C的磷酸化影响其磷酸酶活性。干扰JNK降解或改变其活性可延迟退出有丝分裂并损害DNA损伤后的G2期阻滞。使用生物化学，细胞和遗传模型，我们提出的研究将描绘的要求和调节，这个新确定的调节叶沿着细胞周期和DNA损伤反应。

DESCRIPTION (provided by applicant): JNK (1-3) are members of the stress-activated protein kinase family that regulates the cellular stress response via their control of cell death and survival. Our recent studies disclose an unexpected mechanism for the regulation and consequently the function of JNK; our preliminary results demonstrate that through its KEN-box, JNK is targeted for degradation in a cell cycle-dependent manner. Such targeting is mediated by Cdh1, which recruits the Anaphase Promoting Complex or Cyclosome (APC/C) ubiquitin ligase to ubiquitinate JNK, resulting in its proteasome-dependent degradation. APC/CCdh1-mediated JNK degradation primarily occurs during the exit from mitosis, and G1 phases of the cell cycle. Our preliminary data also identify Cdc25C as JNK substrate at the G2/M-G1 phases of the cell cycle. JNK degradation is important for the regulation of Cdc25C activity and Wee1 stability. Thus, a non-degradable (yet which can be activated) form of JNK (JNKKEN) induces reduced cyclin-B/Cdk1 activity, affects spindle and chromosomal dynamics and delays exit from mitosis. These findings provide the foundation for our hypothesis that through the regulation of Cdc25C, JNK plays an important role in control of cell cycle progression during which it is tightly regulated by the APC/CCdh1. Our finding that JNK is a cell cycle regulator offers an undisclosed link between JNK and unrestrained cell cycle progression as well as chromosome instability, commonly observed in human cancers, where the balance between JNK functions as a stress kinase and cell cycle control protein might be altered. To test our hypothesis we propose to carry out the following studies: Aim 1 - Assess the molecular determinants responsible for JNK activity during the cell cycle and determine which of JNK family members is most important for cell cycle control.; Aim 2- characterize the effect of JNK on Cdc25C as part of its control of cell cycle transition. Aim 3 -Characterize the cytological changes in mitosis that are controlled by JNK and Cdc25C regulation by JNK; Aim 4 - determine the physiological significance of JNK's role in cell cycle control in a JNK-KEN knock-in mouse model. Aim 5 - using genetic, biochemical and cell biology approaches, determine the role of JNK in cell cycle checkpoint pathways following stress and DNA damage and in human melanoma as a model for constitutively deregulated JNK activity. This proposal brings together experts that closely collaborate to address an important and novel function of the key kinase, JNK. This concerted effort will provide the foundation for understanding the role of JNK in cell cycle progression and the implication of its deregulated control to genotoxic stress response as well as to development of human cancer. PUBLIC HEALTH RELEVANCE: This application will test the hypothesis that JNK is important player in cell cycle control prior and more so, in response to DNA damage. Support for this hypothesis comes from studies in which we discovered that JNK is targeted for degradation at G2M-G1 phases of the cell cycle. JNK mediates its function through phosphorylation of Cdc25C which affects its phosphatase activity. Interfering with JNK degradation or altering its activity delays exit from mitosis and impairs G2 arrest after DNA damage. Using biochemical, cellular and genetic models our proposed studies will delineate the requirements and regulation of this newly identified regulatory lobe along the cell cycle and DNA damage response.