Identification of protein kinase A catalytic subunit beta as a novel binding partner of p73 and regulation of p73 function.
Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit beta (PKA-Cbeta) as a novel binding partner of ... p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73alpha associated with PKA-Cbeta in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73alpha. In contrast, p53 failed to bind to PKA-Cbeta. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73alpha-(1-130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cbeta resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73alpha, whereas a kinase-deficient mutant of PKA-Cbeta had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73alpha. Of particular interest, PKA-Cbeta facilitated the intramolecular interaction of p73alpha, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cbeta-mediated inhibitory mechanism of p73 function.
Mesh Terms:
Animals, Apoptosis, Blotting, Western, COS Cells, Catalytic Domain, Cell Cycle Proteins, Cell Line, Cell Line, Tumor, Chromatin Immunoprecipitation, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cyclin-Dependent Kinase Inhibitor p21, DNA Damage, DNA-Binding Proteins, Down-Regulation, Genes, Tumor Suppressor, Glutathione Transferase, Humans, Immunoblotting, Immunoprecipitation, Microscopy, Fluorescence, Models, Genetic, Nuclear Proteins, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Reverse Transcriptase Polymerase Chain Reaction, Subcellular Fractions, Transcriptional Activation, Transfection, Tumor Protein p73, Tumor Suppressor Protein p53, Tumor Suppressor Proteins, Two-Hybrid System Techniques
Animals, Apoptosis, Blotting, Western, COS Cells, Catalytic Domain, Cell Cycle Proteins, Cell Line, Cell Line, Tumor, Chromatin Immunoprecipitation, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cyclin-Dependent Kinase Inhibitor p21, DNA Damage, DNA-Binding Proteins, Down-Regulation, Genes, Tumor Suppressor, Glutathione Transferase, Humans, Immunoblotting, Immunoprecipitation, Microscopy, Fluorescence, Models, Genetic, Nuclear Proteins, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Reverse Transcriptase Polymerase Chain Reaction, Subcellular Fractions, Transcriptional Activation, Transfection, Tumor Protein p73, Tumor Suppressor Protein p53, Tumor Suppressor Proteins, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Apr. 29, 2005
PubMed ID: 15723830
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