Abstract
Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.
Highlights
Fos or ATF family members, c-Jun constitutes the inducible activator protein 1 (AP-1) transcription factor.[1,2] Genetic studies have demonstrated a crucial role for c-Jun/AP-1 in different cellular programs, as cell cycle progression, differentiation and apoptosis.[3,4] c-Jun transactivation is mainly regulated by the c-Jun N-terminal kinase (JNK) pathway, through phosphorylation of its N-terminal domain at S63/S73 and threonine T91/T93.5–9 These two groups of phosphorylation regulate c-Jun transactivity by two distinct mechanisms
We show that abrogation of T91/T93/T95 phosphorylation has no consequence on the capacity of c-Jun to induce neurite outgrowth in PC12 cells, suggesting that site-specific regulation of c-Jun phosphorylation is one possible mechanism underlying the functional dichotomy of the JNK/c-Jun pathway in the brain
To investigate the role of c-Jun phosphorylation at T91/T93 in neuronal death, we set up a previously described experimental system consisting of primary cultures of mouse cerebellar granule cells (CGC) undergoing cell death in response to TK-deprivation.[24]
Summary
Fos or ATF family members, c-Jun constitutes the inducible activator protein 1 (AP-1) transcription factor.[1,2] Genetic studies have demonstrated a crucial role for c-Jun/AP-1 in different cellular programs, as cell cycle progression, differentiation and apoptosis.[3,4] c-Jun transactivation is mainly regulated by the c-Jun N-terminal kinase (JNK) pathway, through phosphorylation of its N-terminal domain at S63/S73 and threonine T91/T93.5–9 These two groups of phosphorylation regulate c-Jun transactivity by two distinct mechanisms. S63/S73 phosphorylation promotes the expression of c-Jun target genes by facilitating c-Jun physical interactions with the coactivator CPB/p300.10,11 In the second mechanism, c-Jun phosphorylation on both These studies suggest that, whereas S63/S73 phosphorylation is necessary for both mechanisms of c-Jun transactivation, T91/T93 phosphorylation may act as a regulatory signal switching ‘on’ specific classes of c-Jun/Ap-1 target genes by transcriptional derepression. In line with this hypothesis, S63/S73 phosphorylation is induced by a myriad of extracellular signals with either transient or prolonged signaling dynamics[14,15] and it functions in diverse physiological pathways, inducing genes involved in cell cycle progression, differentiation and apoptosis.[3,4,16] Differently, T91/T93 phosphorylation by JNK is induced mostly by destructive cues, including genotoxic stress and chemotherapeutic drugs.[8,14,17]. The differential regulation of the S63/S73 sites versus the T91/T93 may reflect a switch-like response of JNK to genotoxic cues, with small stimuli rising S63/S73 phosphorylation and larger supra-threshold stimuli inducing T91/T93 phosphorylation
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