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.
Cells detect and respond to many changes in their environments using a class of enzymes called stress MAP kinases. These enzymes are known to be active under conditions of cellular stress, to function during the normal cell division cycle and to be deregulated in some cancers; for these reasons stress MAP kinases have been targeted for pharmacological inhibition and used as biomarkers in the treatment of pathologies such as inflammatory diseases and cancer. The goal of this research is to understand precisely how MAP kinases function in normal cell proliferation control because the ability to accurately explain and predict the consequences of MAP kinase inhibition for both normal and diseased tissues will substantially improve efforts to treat a wide variety of human diseases.
