title: Identification of protein kinase A catalytic subunit beta as a novel binding partner of p73 and regulation of p73 function.
abstract: 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.
authors.affiliation: Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hanamoto
authors.forename: Toshinori
authors.initials: T
authors.lastname: Ozaki
authors.forename: Kazushige
authors.initials: K
authors.lastname: Furuya
authors.forename: Mitsuchika
authors.initials: M
authors.lastname: Hosoda
authors.forename: Syunji
authors.initials: S
authors.lastname: Hayashi
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Nakanishi
authors.forename: Hideki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Hironobu
authors.initials: H
authors.lastname: Kikuchi
authors.forename: Satoru
authors.initials: S
authors.lastname: Todo
authors.forename: Akira
authors.initials: A
authors.lastname: Nakagawara
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D019556:COS Cells; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D015536:Down-Regulation; D016147:Genes, Tumor Suppressor; D005982:Glutathione Transferase; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D015533:Transcriptional Activation; D014162:Transfection; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D020798:Two-Hybrid System Techniques
chemical_list: C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases