Phosphorylation of c-Jun is necessary for apoptosis induced by survival signal withdrawal in cerebellar granule neurons.

Cerebellar granule neurons die by apoptosis when deprived of survival signals. This death can be blocked by inhibitors of transcription or protein synthesis, suggesting that new gene expression is required. Here we show that c-jun mRNA and protein levels increase rapidly after survival signal withdrawal and that transfection of the neurons with an expression vector for a c-Jun dominant negative mutant protects them against apoptosis. Phosphorylation of serines 63 and 73 in the c-Jun transactivation domain is known to increase c-Jun activity. By using an antibody specific for c-Jun phosphorylated on serine 63, we show that this site is phosphorylated soon after survival signal withdrawal. To determine whether c-Jun phosphorylation is necessary for apoptosis, we have expressed c-Jun phosphorylation site mutants in granule neurons. c-Junasp, a constitutively active c-Jun mutant in which the known and potential serine and threonine phosphoacceptor sites in the transactivation domain have been mutated to aspartic acid, induces apoptosis under all conditions tested. In contrast, c-Junala, which cannot be phosphorylated because the same sites have been mutated to alanine, blocks apoptosis caused by survival signal withdrawal. Finally, we show that cerebellar granule neurons contain high levels of Jun kinase activity and low levels of p38 kinase activity, neither of which increases after survival signal withdrawal. Mitogen-activated protein kinase activity decreases under the same conditions. These results suggest that c-Jun levels and c-Jun phosphorylation may be regulated by novel mechanisms in cerebellar granule neurons.