Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons

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.

Figure 1

c-Jun N-terminal phosphorylation in cerebellar granule neurons. (a) CGCs were cultured in 25 mM K and then switched to 5 mM K medium for 18 h, either in absence or in presence 10 μM SP600125 (SP) or 10 μM LiCl, as indicated. Cells were then fixed and nuclei were stained with DAPI. Condensed nuclei were counted by fluorescence microscopy. Columns indicate the average percentage number of condensed nuclei present in 10 different fields, with bars indicating S.D. (b) Western blot analysis of CGCs cultured either in 25 or 5 mM K medium for 4 h and treated with either SP600125 (10 μM) or LiCl (10 μM) as indicated. c-Jun N-terminal phosphorylation or phosphorylated JNK (p-JNK) were determined by using the indicated antibodies. c-Jun expression levels were measured by using a total c-Jun antibody as indicated. Protein levels were normalized by using the Mcm7 antibody. (c) Densitometric analysis of western blot analysis shown in panel c. Values are presented as optical density (OD; expressed in arbitrary units) relative to the control (Mcm7). Bars represent S.D. from three different densitometric quantification of analogous experiments shown in panel c. (d–f) Immunofluorescence analysis of CGCs cultured in 25 or 5 mM K medium for 4 h and treated with either SP600125 (10 μM) or LiCl (10 μM) as indicated. Cells were then fixed and nuclei were stained with DAPI; c-Jun N-terminal phosphorylation and expression were detected by immunocytochemical staining using c-Jun-p63 Ab (d), or c-Jun-pT-91/T93 (e), or c-Jun total Ab (f). (g) Immunofluorescence analysis of CGC cultures TK-deprived for 18 h and analyzed for condensed nuclei by DAPI staining. (h) Quantification of results shown in d–g. Columns indicate the average percentage number of immunoreactive cells (d–f) or condensed nuclei (g) present in 10 different fields, with bars indicating S.D.

Figure 2

Lack of T91/93 phosphorylation protects from cell death. (a and b) Confocal analysis of CGCs generated from c-Jun-ΔN mice and infected with GFP-tagged lentivirus particles expressing either HA-tagged c-Jun proteins: c-Jun-wt-HA (a) or c-JunA95-HA (b). After infections, cells were cultured for 6 days and then treated as indicated for 18 h. After fixation, nuclei were stained by DAPI and immunostained with HA antibody. (c) Quantification of results shown in a and b. Columns indicate the average percentage number of condensed nuclei present in 10 different fields, with bars indicating S.D. (d) Western blot analysis of CGCs from c-Jun-ΔN mice infected with lentivirus as described in a and b. After infection, cells were cultured either in 25 mM K or shifted to 5 mM K medium for 4 h, as indicated. Control represents not infected CGCs. c-Jun N-terminal phosphorylation was determined by using the indicated c-Jun phospho-specific antibodies. The levels of c-Jun-HA proteins were determined by using the HA antibody. (e) CGCs from c-Jun-ΔN mice were transfected with Jun2-luc reporter plasmids in combination with plasmids expressing either c-Jun-wt-HA or c-JunA95-HA proteins or empty vector (indicated as control). After infection, cells were cultured either in 25 mM K or shifted to 5 mM K medium for 4 h, as indicated. The luciferase activities were normalized to the internal transfection control. Values of CGCs transfected with empty vectors and cultured in 25 mM K were set as onefold induction. Bar graphs represent the mean ±S.D. of three independent assays

Figure 3

Lack of T91/93 phosphorylation does not impair neurite outgrowth. (a) Confocal analysis of PC12 cells were cultured either in absence or in presence of NFG (72 h) as indicated. After fixation, cells were immunostained with neurofilament (NLF, green) antibodies. Nuclei were stained with DAPI (b) Naive PC12 cells were transfected with DNA constructs expressing either HA-tagged c-Jun-wt or Ha-tagged c-JunA95 constructs, as indicated. Transfected cells were cultured in absence of NGF for 72 h; after fixation cells were incubated with either HA (red) or NLF, green antibodies. (c) Columns indicates the average percentage number of PC12 cells with neurites present in 10 different fields, with bars indicating S.D.

Figure 4

Deregulation of T91/T93 phosphorylation recovers cell death in presence of lithium. (a and b) Confocal analysis of CGCs generated from c-Jun ΔN mice and infected with GFP-tagged lentivirus particles expressing c-Jun-wt-HA (a) or Ha-c-JunD95-HA proteins (b). After transduction, cells were cultured for 6 days and then treated as indicated with 25 or 5 mM K for 18 h, with or without SP600125 (10 μM) or LiCl (10 μM) as indicated. After fixation, nuclei were stained by DAPI and immunostained with HA antibody (red). (c) Quantification of results shown in a and b. Columns indicate the average percentage number of condensed nuclei present in 10 different fields, with bars indicating S.D.

Figure 5

Phosphorylation of amino-acid residues within c-Jun N-terminal domain. (a) Western blot analysis of HEK-293 cells transfected with DNA constructs expressing either HA-tagged c-Jun-wt or Ha-tagged c-JunA95, as indicated. After transfection, cells were treated either without or with Anisomycin (100 ng/ml) for indicated time-periods. c-Jun N-terminal phosphorylation was determined by using the indicated antibodies. (b) Lysates from anisomycin-treated c-Jun-wt-transfected HEK-T cells were immunoprecipited using the c-total c-Jun antibody, then aliquots from c-Jun immunocomplexes were analyzed by western blot using either c-Jun total antibodies (left panel) or c-Jun-p91/93 antibodies (right panel). (c) MALDI MS/MS spectra of the [M+H]⁺ from segments 82–97 carrying three phosphate groups at T91, T93 and T95. (d) MALDI MS/MS spectra of the [M+Na]⁺ from segments 62–70 carrying one phosphate group at S63. The a, b and y ions are labeled in each spectrum, as well as b or y ions corresponding to the neutral loss of phosphoric acid (−98)

Figure 6

JNK-1 phosphorylates in vitro c-Jun at T91/93 in a T95-dependent manner. (a) NSC-34 cells were transfected with plasmids expressing either c-Jun-wt-HA or c-JunA95-HA-tagged proteins (as indicated) and levels of c-Jun site-specific phosphorylation were analyzed by western blot of total extracts by using the indicated antibodies. (b) Either c-Jun-wt-HA or c-JunA95-HA-tagged proteins were immunoprecipitated by using HA antibodies, then c-Jun-HA immunocomplexes were used as substrates for in vitro phosphorylation assays by using recombinant JNK-1, either alone or in combination with recombinant GSK3β as indicated. Products of in vitro phosphorylation reactions were analyzed by western blot using c-Jun-phospho-specific antibodies for either p-T91/T93, p-S63 or p-239. Total amounts of c-Jun proteins were determined by using the c-Jun total antibody. (c) In vitro phosphorylation assay of recombinant FL-c-Jun protein (Ezio Life Sciences) incubated with recombinant JNK-1 as indicated. Reactions were analyzed by western blot, using either c-Jun-p91/93 or c-Jun-p-S63 phospho-specific antibodies as indicated. Total amounts of c-Jun proteins were determined by using the c-Jun total antibody