title: p53 binding prevents phosphatase-mediated inactivation of diphosphorylated c-Jun N-terminal kinase.
abstract: c-Jun N-terminal kinase (JNK) is a serine/threonine phosphotransferase whose sustained activation in response to genotoxic stress promotes apoptosis. In Drosophila, the normally rapid JNK-dependent apoptotic response to genotoxic stress is significantly delayed in Dmp53 (Drosophila p53) mutants. Likewise, the extent of JNK activity after UV irradiation is dependent on p53 in murine embryonic fibroblasts with loss of p53 resulting in diminished JNK activity. Together, these results suggest that p53 potentiates the JNK-dependent response to genotoxic stress; however, the mechanism whereby p53 stimulates JNK activity remains undefined. Here, we demonstrate that both Drosophila and human p53 can directly stimulate JNK activity independently of p53-dependent gene transcription. Furthermore, we demonstrate that both the Drosophila and human p53 orthologs form a physical complex with diphosphorylated JNK ((DP)JNK) both in vivo and in vitro, suggesting that the interaction is evolutionarily conserved. Focusing on human p53, we demonstrate that the interaction maps to the DNA binding domain (hp53(DBD)). Intriguingly, binding of p53(DBD) alone to (DP)JNK prevented its inactivation by MAPK phosphatase (MKP)-5; however, JNK was still able to phosphorylate c-Jun while in a complex with the p53(DBD). Apparent dissociation constants for the p53(DBD)·(DP)JNK (274 ± 14 nm) and MKP-5·(DP)JNK (55 ± 8 nm) complexes were established; however, binding of MKP-5 and p53 to JNK was not mutually exclusive. Together, these results suggest that stress-dependent increases in p53 levels potentiate JNK activation by preventing its rapid dephosphorylation by MKPs and that the simultaneous activation of p53 and JNK may constitute a "fail-safe" switch for the JNK-dependent apoptotic response.
authors.affiliation: Departments of Biochemistry and The University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Pramod S
authors.initials: PS
authors.lastname: Gowda
authors.affiliation: Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, Texas 78229; Department of Pediatrics and The University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Fuchun
authors.initials: F
authors.lastname: Zhou
authors.affiliation: Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Linda V
authors.initials: LV
authors.lastname: Chadwell
authors.affiliation: Departments of Biochemistry and The University of Texas Health Science Center, San Antonio, Texas 78229; Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, Texas 78229. Electronic address: mcewen@uthscsa.edu.
authors.forename: Donald G
authors.initials: DG
authors.lastname: McEwen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D046912:Multiprotein Complexes; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; C120407:DUSP10 protein, human; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054637:Dual-Specificity Phosphatases