ABSTRACT
The intra-cellular level of tumor suppressor protein p53 is tightly controlled by an autoregulatory feedback loop between the protein and its negative regulator MDM2. The role of MDM2 in down-regulating the p53 response in unstressed conditions and in the post-stress recovery phase is well documented. However, interplay between the N-terminal phosphorylations and C-terminal acetylations of p53 in this context remains unclear. Here, we show that an MAR binding protein SMAR1 interacts with MDM2 and the Ser15 phosphorylated form of p53, forming a ternary complex in the post stress-recovery phase. This triple complex formation between p53, MDM2 and SMAR1 results in recruitment of HDAC1 to deacetylate p53. The deacetylated p53 binds poorly to the target promoter (p21), which results in switching off the p53 response, essential for re-entry into the cell cycle. Interestingly, the knock-down of SMAR1 using siRNA leads to a prolonged cell-cycle arrest in the post stress recovery phase due to ablation of p53-MDM2-HDAC1 interaction. Thus, the results presented here for the first time highlight the role of SMAR1 in masking the active phosphorylation site of p53, enabling the deacetylation of p53 by HDAC1-MDM2 complex, thereby regulating the p53 transcriptional response during stress rescue.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Transcription, Genetic , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Acetylation , Animals , Cell Cycle Proteins/genetics , Cell Line , DNA/metabolism , DNA Damage , DNA-Binding Proteins/genetics , Gene Expression Regulation , Histone Deacetylase 1 , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Multiprotein Complexes , Nuclear Proteins/genetics , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-mdm2/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
Histone acetyltransferases form crucial links in transducing extrinsic signals to actual initiation of transcription. A multitude of stress signal integrations occur through the interaction of p300 with p53 phosphorylated at different residues of the transactivation domain. How such interactions activate different gene expression programs remains largely unknown. p300 contains at least five domains that are known to interact with p53, but their role in transcription regulation is not known. We measured the binding affinity of various phosphorylated transactivation domains towards several p53 binding domains of p300 by fluorescence anisotropy. The binding affinities of different phosphorylated transactivation domains of p53 towards different domains of p300 vary by several orders of magnitude, indicating that interactions of different post-translationally modified forms of p53 may occur through different domains of p300. Thus, different post-translationally modified p53 fragments may form transcription-initiating complexes of different configurations, leading to the activation of different promoters and pathways.
Subject(s)
Protein Interaction Domains and Motifs , Tumor Suppressor Protein p53/metabolism , p300-CBP Transcription Factors/metabolism , Fluorescence Polarization , Protein Binding , Tumor Suppressor Protein p53/chemistry , p300-CBP Transcription Factors/chemistryABSTRACT
Several phosphorylations are known to occur in the N-terminal transactivation domain of human p53. To explore the structural effects of these phosphorylations, we have chemically synthesized the unphosphorylated p53-(1-39) and its three phosphorylated analogs, phosphorylated at Ser-15, Thr-18, and Ser-20. p53-(1-39) and its Ser-15 and Thr-18 phosphorylated analogs were tested for interaction with p300. The order of binding affinities was similar to that derived from biochemical experiments with the whole protein, indicating functional integrity of the domain. Differences in chemical shifts and coupling constants indicate significant structural changes upon phosphorylations. The single tryptophan in the unphosphorylated domain has an emission maximum and a Stern-Volmer constant that are characteristics of tryptophans situated in protein interiors. The diffusion constant is monomer-like, with an axial ratio of 1:7.5, indicating a significant degree of compaction. Upon phosphorylations, the emission maximum and diffusion constant change significantly toward values that indicate more open conformations. Binding of the hydrophobic probe bis-1-anilino-8-naphthalenesulfonate to the unphosphorylated and one of the phosphorylated domains is also significantly different, suggesting different conformations. We propose that phosphorylations switch the largely folded transactivation domain to more open conformations that interact with transcription factors such as p300/cAMP- responsive element-binding protein-binding protein, leading to enhancement of gene expression.
