| Summary: | 博士 === 國立臺灣大學 === 分子醫學研究所 === 93 === Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase. Its functions in pro-apoptosis and tumor suppression have been studied before, but detailed mechanism isn’t fully elucidated. In the first part of the study, we demonstrate that DAPK is capable of phosphorylating the regulatory light chain of myosin II (MLC) at serine 19 in vitro and in vivo, resulting in stress fibers stabilization. However, DAPK cannot stimulate the formation of focal adhesion in quiescent cells and promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Thus, we proposed that DAPK functions in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity. In the second part of the study, we focus on the mechanism through which DAPK functions as a tumor suppressor. DAPK is thought to execute tumor suppressive function by its apoptotic activity. However, the apoptotic effect of DAPK is largely p53-dependent, while many tumor cells are p53 defective. To reconcile this, we tested whether DAPK has another mechanism to suppress tumor. As DAPK has been demonstrated to the regulate cytoskeleton and integrin activity, both of which play a role in cell migration, we therefore study the function of DAPK in migration. We found that DAPK inhibits random migration by reducing directional persistence and directed migration by blocking cell polarization. These DAPK-mediated migratory defects are mainly through its suppression of integrin/Cdc42 pathway. The regulation of migration by DAPK indeed in part explains for its tumor suppressor function, exemplified by the fact that in certain p53-mutant tumor cells that are resistant to DAPK-induced apoptosis, DAPK expression can still block their migratory and invasive abilities. Furthermore, by using a paired of lung adenocarcinoma cell lines, which are of the same source but of different invasive activities, we demonstrated that DAPK expression level is a determinant factor in tumor invasiveness. There emerges the second, p53-independent, mechanism for DAPK in tumor suppression. To sum up, to get insight how DAPK exerts its physiological function, we demonstrate that DAPK uncouples stress fibers and focal adhesions, partly through its phosphorylation of MLC. Besides, we uncover a new p53-independent mechanism mediated by DAPK to affect cell motility, accounting for its tumor suppressive functions.
|
|---|
