ABSTRACT
Akt/protein kinase B is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway and plays a critical role in promotion of cell survival. The function of transcriptional coactivator p300 is required by many transcription factors to either activate or repress gene expression. Here, we show that induction of PI3K enhances the metabolic stability of endogenous p300 protein. On the other hand, repression of PI3K by LY294002 induces p300 degradation through the 26S proteasome pathway and impedes the transcriptional activity of the coactivator. In addition, Akt interacts with the coactivator and the activity of Akt is required to maintain the steady-state level of p300. Our study provides a new insight into the molecular mechanisms by which the critical concentration of p300 protein is regulated and suggests a role for Akt in control of various cellular activities through the transcriptional coactivator p300.
Subject(s)
Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Acetyltransferases/chemistry , Acetyltransferases/genetics , Acetyltransferases/metabolism , Cell Line, Tumor , Chromones/pharmacology , Cycloheximide/pharmacology , Enzyme Stability , Histone Acetyltransferases , Humans , Morpholines/pharmacology , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Peptide Hydrolases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Trans-Activators/chemistry , Trans-Activators/geneticsABSTRACT
To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.
