Integrating stress MAP kinase signaling with DNA replication origin licensing

描述(由申请人提供)：及时、完整和精确的DNA复制和准确的染色体分离对于控制细胞增殖和基因组稳定性至关重要。控制不当的复制会导致细胞死亡、组织退化、 和癌症。出于这些原因，我们试图定义管理DNA复制能力和与细胞周期控制协调的分子机制。起源许可是DNA复制的最早步骤，如果不能协调起源许可与细胞分裂周期，就会导致复制不足或重新复制的染色体区域，这是细胞死亡和基因组不稳定的来源。我们的长期目标是准确地确定来源许可蛋白是如何受到细胞内细胞周期信号和细胞外信号通路的调控的。目前的范例依赖于通过细胞周期蛋白依赖性蛋白激酶(CDK)对起源许可的直接和间接控制相结合。根据已有文献记载的在细胞增殖中的作用，我们推测，由应激激活的MAP激酶p38和JNK介导的信号通路可能对原产地许可具有同样重要的影响。最近的研究表明，即使在不受干扰的细胞周期中，应激蛋白激酶也发挥着关键作用，但它与细胞周期中的基本事件之间的界面还不完全清楚。我们的研究表明，这些应激映射蛋白激酶直接抑制来源许可，并通过直接磷酸化使许可蛋白CDT1失活。重要的是，其他人已经表明，应激映射蛋白激酶也在不受干扰的细胞周期中发挥作用，以控制细胞静止、G2期和有丝分裂--在这些正常情况下，必须抑制起源许可。确定MAP激酶抑制起源许可的机制是这一提议的重点之一。此外，我们还发现了CDT1的一种不依赖于其复制作用的新的染色体分离功能，并且MAP激酶介导的磷酸化影响了这种CDT1有丝分裂功能。我们假设，应激映射蛋白直接控制染色体复制能力和有丝分裂分离的基本步骤。我们的目标是确定MAP激酶调节原产地许可蛋白的机制和生理作用。结合这一目标，我们以前的研究和专业知识启发了以下目标：(1)确定应激图激酶在阻止细胞起源许可和CDT1功能中的作用，(2)确定应激图激酶在细胞周期退出时建立静止细胞的无许可状态中的作用，(3)确定MAP激酶介导的CDT1磷酸化在调节CDT1‘S功能中在染色体分离中的作用。阐明p38和JNK映射激酶影响这些关键细胞周期转变的机制将导致新的、集成的细胞周期控制模型。然后，这些模型可以用来理解在正常细胞增殖、分化和组织动态平衡以及在慢性炎症和癌症等病理环境中激活或抑制应激映射激酶的生理后果。

DESCRIPTION (provided by applicant): Timely, complete, and precise DNA replication followed by accurate chromosome segregation is critical for controlling cell proliferation and genome stability. Improperly controlled replication contributes to cell death, tissue degeneration, and cancer. For these reasons we seek to define molecular mechanisms that govern DNA replication competence and coordination with cell cycle control. Origin licensing is the earliest step in DNA replication, and failure to coordinate origin licensing with the cell division cycle leads to under-replicated or re- replicated chromosomal regions which are sources of cell death and genome instability. Our long-term goal is to determine precisely how origin licensing proteins are regulated by intracellular cell cycle cues and by extracellular signaling pathways. Current paradigms rely on a combination of direct and indirect control of origin licensing by the cyclin dependent kinases (CDKs). Based on documented roles in cell proliferation, we reasoned that a signaling pathway mediated by the stress-activated MAP kinases, p38 and JNK, could have an equally important impact on origin licensing. Recent studies have shown that stress MAP kinases also play key roles even in unperturbed cell cycles, but the interfaces between MAP kinases and fundamental events in the cell cycle are not fully known. Our investigations revealed that these stress MAP kinases directly inhibit origin licensing, and they inactivate the licensing protein Cdt1 by direct phosphorylation. Importantly, others have shown that stress MAP kinases also function in unperturbed cell cycles to govern cellular quiescence, G2 phase, and mitosis - normal circumstances in which origin licensing must be inhibited. Determining the mechanisms of origin licensing inhibition by MAP kinases is one focus of this proposal. In addition, we have uncovered a novel chromosome segregation function for Cdt1 independent of its replication role, and MAP kinase-mediated phosphorylation affects this Cdt1 mitotic function. We hypothesize that stress MAP kinases directly govern fundamental steps in chromosome replication competence and mitotic segregation. Our objective is to determine the mechanisms and physiological roles of MAP kinase regulation of origin licensing proteins. Our prior studies and expertise coupled with this objective inspire the following Aims: (1) Determine the role of stress MAP kinases in blocking origin licensing and Cdt1 function in proliferating cells, (2) Determine the role of stress MAP kinases in establishing the unlicensed state of quiescent cells during cell cycle exit, (3) Determine the role of MAP kinase-mediated Cdt1 phosphorylation in regulating Cdt1's function in chromosome segregation. Elucidating the mechanisms by which p38 and JNK MAP kinases impact these key cell cycle transitions will lead to new, integrated models of cell cycle control. These models can then be used to understand the physiological consequences of activating or inhibiting the stress MAP kinases during normal cell proliferation, differentiation, and organismal homeostasis and in pathological settings such as chronic inflammation and cancer.