title: Negative charged threonine 95 of c-Jun is essential for c-Jun N-terminal kinase-dependent phosphorylation of threonine 91/93 and stress-induced c-Jun biological activity.
abstract: Activation of c-Jun, a major component of the AP-1 transcription factor, represents a paradigm for transcriptional response to stress. Transactivation of c-Jun is regulated by Jun-N-terminal kinases (JNKs) through phosphorylation at serine 63 and 73 (S63/S73), as well as at threonine 91 and 93 (T91/T93). How these two groups of phosphoacceptor sites respond to different grades of genotoxic stress and whether DNA-damage pathways influence the extent of their JNK-dependent phosphorylations remain to be elucidated. Here, we show that following a short exposure to the DNA-damaging compound etoposide, c-Jun phosphorylation is restricted to S63/S73. In contrast, JNK-dependent phosphorylation of T91/T93 requires continuous exposure to the drug and is impaired by caffeine treatment or alanine substitution of the adjacent threonine 95 (T95). Conversely, c-Jun mutations switching the T95/Q96 site into a canonical site for mitogen activated protein kinase (MAPK) phosphorylation (T95/P96) rescues T91/T93 phosphorylation in presence of caffeine, suggesting that a preceding phosphorylation at T95 exposes T91/T93 to JNK-dependent phosphorylation. Moreover, we show that alanine substitution at T95 impairs c-Jun transactivation and c-Jun-mediated cell death, indicating that negatively charged T95 is a general constraint for c-Jun activation. Hence, our study suggests that c-Jun may sense the strength of genotoxic stress through DNA-damage dependent phosphorylation of T95, which in turn augments c-Jun transactivation by JNKs.
authors.affiliation: Dipartimento Farmaco-biologico, Universita' della Calabria, 87036 Rende (CS), Italy. kachera@libero.it
authors.forename: Maria
authors.initials: M
authors.lastname: Vinciguerra
authors.forename: Ilaria
authors.initials: I
authors.lastname: Esposito
authors.forename: Salvatore
authors.initials: S
authors.lastname: Salzano
authors.forename: Antonio
authors.initials: A
authors.lastname: Madeo
authors.forename: Georg
authors.initials: G
authors.lastname: Nagel
authors.forename: Marcello
authors.initials: M
authors.lastname: Maggiolini
authors.forename: Adriana
authors.initials: A
authors.lastname: Gallo
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Musti
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013912:Threonine / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D015533:Transcriptional Activation
chemical_list: D000838:Anions; D016755:Proto-Oncogene Proteins c-jun; D013912:Threonine; D001224:Aspartic Acid; D048031:JNK Mitogen-Activated Protein Kinases