| Glycogen synthase kinase-3β (GSK3β) is a central figure in many signaling pathways, including the wnt pathway, and is an important proapoptotic signaling enzyme. This study found that DNA damage increased GSK3β activity, a novel effect of DNA damage. GSK3β directly interacted with the major tumor suppressor protein p53, which accumulated after DNA damage. Overexpression of p53 was sufficient to increase GSK3β activity. Moreover, the in vitro activity of GSK3β was increased in the presence of recombinant p53, indicating that p53 is capable of directly activating GSK3β. p53 domain deletion constructs revealed two regions of p53, the activation domain 1 (AD1) and C-terminal basic domain (BD), that regulated its binding to GSK3β. Deletion of the AD1, but not mutations that prevented p53 binding to MDM2 through the AD1, enhanced GSK3β binding to p53, indicating that the AD1 interferes with the binding of GSK3β to p53. In contrast to the AD1, deletion of the BD of p53 abrogated GSK3β binding, indicating that this region is necessary for binding to GSK3β. p53 is a nuclear protein that functions as a transcription factor and regulates a variety of target genes, such as p21, MDM2, and Bax. Inhibition of GSK3β attenuated p53-induced increases in the expression of p21 and MDM2, indicating that GSK3β facilitates p53 transactivation. GSK3β also facilitated apoptosis mediated by p53 following DNA damage, as pharmacological and molecular inhibitors of GSK3β attenuated DNA damage-mediated activation of caspases-3, a key enzyme in apoptotic signaling. Part of this protection from apoptosis was centered in the mitochondria, where GSK3β activity was found to be necessary for cytochrome c release preceding caspases-3 activation. Therefore, GSK3β and p53 act synergistically to regulate cellular responses to DNA damage, as p53 stimulates GSK3β and GSK3β promotes p53 actions in the nucleus (transcription) and mitochondria. Taken together, these results defined p53 as a novel protein that binds and regulates GSK3β, defined a new regulatory mechanism controlling the actions of p53, defined the region of p53 interacting with GSK3β, and revealed facilitory roles of GSK3β on nuclear and mitochondria actions of p53. |
