LASTING N-TERMINAL PHOSPHORYLATION OF C-JUN AND ACTIVATION OF C-JUN N-TERMINAL KINASES AFTER NEURONAL INJURY

Transcription factor c-Jun is proposed to control neuronal cell death and survival, but its activation by N-terminal phosphorylation and the underlying activity of the c-Jun N-terminal kinases (JNKs) remain to be elucidated in the adult mammalian brain. We generated a polyclonal antiserum that specifically recognizes c-Jun phosphorylated at its ser ine 73 (S73) residue after UV irradiation of 3T3 cells. Disruption of the c-jun locus in 3T3 cells abolished this reaction, and retransfecti on of the human c-jun at the c-jun-/- background restored it. The phos pho-c-Jun antiserum was used to visualize N-terminally phosphorylated c-Jun in the adult rat brain with cellular resolution. Prolonged c-Jun S73 phosphorylation was detected in affected neurons up to 5 d after transient occlusion of medial cerebral artery or up to 50 d after tran section of central nerve fiber tracts. After cerebral ischemia-reperfu sion, phosphorylation of c-Jun was linked with induced expression of F as-ligand (APO-I, CD95-ligand), whose gene is a putative c-Jun/AP-1 ta rget, and with terminal deoxynucleotidyl transferase-mediated biotinyl ated UTP nick end labeling (TUNEL) reactivity, a marker for apoptosis. After nerve fiber transection, however, lasting c-Jun phosphorylation occurred in axotomized neurons negative for Fas-ligand or TUNEL and r egardless of degeneration or survival. In contrast to these lasting ph osphorylation patterns, transient seizure activity by pentylenetetrazo le provoked only a brief c-Jun phosphorylation and JNK activation. In extracts from ischemic or axotomized brain compartments, c-Jun phospho rylation correlated with enhanced longterm JNK activity, and in-gel ki nase assays visualized proteins with sizes corresponding to JNK isofor ms as the only c-Jun N-terminally phosphorylating enzymes. These resul ts demonstrate that lasting c-Jun S73 phosphorylation and JNK activity are part of neuronal stress response after neurodegenerative disorder s in the adult mammalian brain with Fas-ligand as a putative apoptotic effector.