Characterization of JNK in Cell Cycle Control

描述(申请人提供)：JNK(1-3)是压力激活蛋白激酶家族的成员，通过控制细胞的死亡和存活来调节细胞的应激反应。我们最近的研究揭示了一种意想不到的调节机制，从而揭示了JNK的功能；我们的初步结果表明，通过其Ken-box，JNK以细胞周期依赖的方式被降解。这种靶向是由CDH1介导的，它招募后期促进复合体或环体(APC/C)泛素连接酶泛素化JNK，导致其蛋白酶体依赖的降解。APC/CCdh1介导的JNK降解主要发生在细胞有丝分裂退出和细胞周期的G1期。我们的初步数据还确定，在细胞周期的G2/M-G1期，CDC25C是JNK底物。JNK的降解对于调节CDC25C的活性和Wee1的稳定性很重要。因此，一种不可降解(但可以激活)形式的JNK(JNKKEN)会导致细胞周期蛋白B/CDK1活性降低，影响纺锤体和染色体的动力学，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，即通过对CDc25C的调控，JNK在控制细胞周期进程中发挥着重要作用，在此过程中，它受到APC/CCdh1的严格调控。我们的发现是，JNK是一种细胞周期调节因子，在JNK和不受限制的细胞周期进展以及染色体不稳定之间提供了一种未披露的联系，这在人类癌症中很常见，在这种情况下，JNK作为应激蛋白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JNK活性的分子决定因素，并确定哪些JNK家族成员对细胞周期控制最重要。目的2-表征JNK对CDC25C的影响，作为其控制细胞周期转换的一部分。目的3-研究JNK和CDC25C调控有丝分裂的细胞学变化；目的4-确定JNK在JNK-KEN基因敲入小鼠模型中细胞周期调控中的生理学意义。目的5-利用遗传学、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通路中的作用，以及在人类黑色素瘤中作为结构性去调控JNK活性的模型。这项提议将密切合作的专家聚集在一起，以解决关键激酶JNK的一个重要和新的功能。这一共同努力将为理解JNK在细胞周期进程中的作用以及其对遗传毒性应激反应和人类癌症发展的非调控的影响奠定基础。公共卫生相关性：这项应用将检验JNK在细胞周期控制中的重要作用这一假设，尤其是在应对DNA损伤方面。支持这一假设的研究中，我们发现JNK的目标是在细胞周期的G2M-G1期降解。JNK通过CDC25C的磷酸化来调节其功能，而CDC25C则影响其磷酸酶活性。干扰JNK的降解或改变其活性会延迟有丝分裂的退出，并损害DNA损伤后的G2停滞。利用生化、细胞和遗传模型，我们的研究将描绘出这个新发现的调节叶在细胞周期和DNA损伤反应中的要求和调控。

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.