Integrating stress MAP kinase signaling with DNA replication origin licensing

描述（由申请方提供）：及时、完整和精确的DNA复制，随后进行准确的染色体分离，对于控制细胞增殖和基因组稳定性至关重要。不适当的复制控制会导致细胞死亡，组织退化， 和癌症由于这些原因，我们试图定义的分子机制，管理DNA复制能力和协调与细胞周期控制。来源许可是DNA复制中的最早步骤，并且未能协调来源许可与细胞分裂周期导致复制不足或再复制的染色体区域，其是细胞死亡和基因组不稳定性的来源。我们的长期目标是精确地确定起源许可蛋白是如何被细胞内细胞周期信号和细胞外信号通路调节的。目前的模式依赖于细胞周期蛋白依赖性激酶（CDK）对来源许可的直接和间接控制的组合。基于在细胞增殖中记录的作用，我们推断由应激激活的MAP激酶p38和JNK介导的信号通路可能对来源许可产生同样重要的影响。最近的研究表明，即使在未受干扰的细胞周期中，应激MAP激酶也起着关键作用，但MAP激酶与细胞周期中的基本事件之间的界面尚不完全清楚。我们的研究表明，这些应激MAP激酶直接抑制起源许可，它们通过直接磷酸化来抑制许可蛋白Cdt 1。重要的是，其他人已经表明，应激MAP激酶也在未受干扰的细胞周期中起作用，以控制细胞静止、G2期和有丝分裂-其中起源许可必须被抑制的正常情况。确定MAP激酶的起源许可抑制机制是本建议的重点之一。此外，我们已经发现了一种新的染色体分离功能Cdt 1独立的复制作用，和MAP激酶介导的磷酸化影响Cdt 1有丝分裂功能。我们推测，压力MAP激酶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激酶调节的起源许可蛋白的机制和生理作用。我们之前的研究和专业知识与这一目标相结合，激发了以下目标：（1）确定应激MAP激酶在阻断增殖细胞中的起源许可和Cdt 1功能中的作用，（2）确定应激MAP激酶在细胞周期退出期间建立静止细胞的未许可状态中的作用，（3）研究MAP激酶介导的Cdt 1磷酸化在调节Cdt 1在染色体分离中的作用。阐明p38和JNK MAP激酶影响这些关键细胞周期转换的机制将导致新的细胞周期控制的综合模型。然后，这些模型可用于理解在正常细胞增殖、分化和生物体内平衡期间以及在病理环境如慢性炎症和癌症中激活或抑制应激MAP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.