ABSTRACT
Ataxia-telangiectasia mutated (ATM) kinase is a component of a signalling mechanism that determines the process of decision-making in response to DNA damage and involves the participation of multiple proteins. ATM is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and orchestrates signalling cascades that initiate the DNA damage response. Cells lacking ATM are hypersensitive to insults, particularly genotoxic stress, induced through radiation or radiomimetic drugs. Here, we investigate the degree of ATM activation during time-dependent treatment with genotoxic agents and the effects of ATM on phospho-induction and localization of its downstream substrates. Additionally, we have demonstrated a new cell-cycle-independent mechanism of ATM gene regulation following ATM kinase inhibition with KU5593. Inhibition of ATM activity causes induction of ATM protein followed by oscillation and this mechanism is governed at the transcriptional level. Furthermore, this autoregulatory induction of ATM is also accompanied by a transient upregulation of p53, pATR and E2F1 levels. Since ATM inhibition is believed to sensitize cancer cells to genotoxic agents, this novel insight into the mechanism of ATM regulation might be useful for designing more precise strategies for modulation of ATM activity in cancer therapy.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/drug effects , Enzyme Inhibitors/pharmacology , Morpholines/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/drug effects , Pyrones/pharmacology , Transcriptional Activation/drug effects , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/drug effects , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Ataxia Telangiectasia Mutated Proteins , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Cell Line, Tumor , Cells, Cultured , DNA Damage , DNA-Binding Proteins/genetics , E2F1 Transcription Factor/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Up-RegulationABSTRACT
Cyclin-dependent protein kinases play important roles in cell cycle progression and are attractive targets for the design of anti-proliferative drugs. Two distinct synthetic CDK1/2 inhibitors, Roscovitine and NU2058, are pharmacologically distinct in their ability to modify p53-dependent transcription and perturb cell cycle progression. Although such active-site CDK1/2 inhibitors comprise the most standard type of enzyme inhibitor, many protein kinases are proving to harbour high affinity docking sites that may provide a potentially novel interface for the design of kinase-inhibitors. We examined whether CDK2 has a docking site for its oligomeric substrate p53, whether small-peptide leads can be developed that inhibit CDK2 function, and whether such peptide-inhibitors are pharmacologically distinct from Roscovitine or NU2058. A docking site for CDK2 was identified in the tetramerization domain of p53 at a site that is distinct from the phospho-acceptor site. Peptides derived from the tetramerization domain of p53 block CDK2 phosphorylation and identification of critical CDK2 contacts in the tetramerization domain of p53 suggest that kinase docking does not require tetramerization of the substrate. Transient transfection assays were developed to show that the GFP-CDK2 docking site fusion protein (GFP-CIP) attenuates p53 activity in vivo and suppresses p21WAF1 induction which is similar to NU2058 but distinct from Roscovitine. A stable cell line with an inducible GFP-CIP gene attenuates p53 activity and induces significant cell death in a drug-resistant melanoma cell line, sensitizes cells to death induced by Doxorubicin, and suppresses cell growth in a colony formation assay. These data indicate that CDK2, in addition to cyclin A, can have a high affinity docking site for a substrate and highlights the possibility that CDK2 docking sites may represent effective targets for inhibitor design.
