title: Comparison between the timing of JNK activation, c-Jun phosphorylation, and onset of death commitment in sympathetic neurones.
abstract: We have investigated the relationship between c-Jun N-terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time-dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three- to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4-(8-chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (> or = 14 h), although this also resulted in near-complete suppression of total JNK activity and short-term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c-Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c-Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic-factor deprivation was also associated with enhanced phosphorylation of c-Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho-c-Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c-Jun phosphorylation are not necessarily rate-limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c-Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c-Jun mediates death by increasing the expression of "death" genes, these must become effective very close to the death commitment point.
authors.affiliation: Department of Biochemistry, University of Cambridge, England, U.K.
authors.forename: K
authors.initials: K
authors.lastname: Virdee
authors.forename: A J
authors.initials: AJ
authors.lastname: Bannister
authors.forename: S P
authors.initials: SP
authors.lastname: Hunt
authors.forename: A M
authors.initials: AM
authors.lastname: Tolkovsky
mesh_terms: D000818:Animals; D017209:Apoptosis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases; D009414:Nerve Growth Factors / Q000008:administration & dosage / Q000494:pharmacology; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D051381:Rats; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D009414:Nerve Growth Factors; D016755:Proto-Oncogene Proteins c-jun; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases