Characterization of the c-Jun N-terminal kinase-BimEL signaling pathway in neuronal apoptosis

Abstract

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in the nervous system; however, the mechanisms by which JNK triggers neuronal apoptosis remain incompletely understood. Recent studies suggest that in addition to inducing transcription of pro-apoptotic genes, JNK also directly activates the cell death machinery. Here, we report that JNK catalyzed the phosphorylation of the BH3-only protein Bcl-2 interacting mediator of cell death (BimEL) at serine 65, both in vitro and in vivo. The JNK-induced phosphorylation of BimEL at serine 65 promoted the apoptotic effect of BimEL in primary cerebellar granule neurons. We also characterized the role of the JNK-BimEL signaling pathway in apoptosis that was triggered by overexpression of the p75 neurotrophin receptor (p75NTR). We found that activation of p75NTR induced the JNK-dependent phosphorylation of endogenous BimEL at serine 65 in cells. The genetic knockdown of BimEL by RNA interference or the expression of a dominant interfering form of BimEL significantly impaired the ability of activated p75NTR to induce apoptosis. Together, these results suggest that JNK-induced phosphorylation of BimEL at serine 65 mediates p75NTR-induced apoptosis. Our findings define a novel mechanism by which a death-receptor pathway directly activates the mitochondrial apoptotic machinery.

Figure 2.

JNK phosphorylates Bim_EL at the distinct site of serine 65 in vitro. A, Recombinant GST-Bim_EL (WT) and recombinant Bim_EL phosphorylation mutants GST-Bim_ELS55A (55A), GST-Bim_ELS65A (65A), and GST-Bim_ELT112A (112A) were incubated with no kinase or recombinant JNK1α1 and subjected to an in vitro kinase assay using [γ-³²P]ATP. Phosphorylated substrates were separated by SDS-PAGE, stained with Coomassie blue (bottom panel), and analyzed by autoradiography (top panel). Degradation products of bacterially expressed GST-Bim_EL are indicated with an asterisk. B, Recombinant GST, GST-Bim_EL (WT), and GST-Bim_ELS65A (65A) were subjected to an in vitro kinase assay with recombinant JNK1α1. Phosphorylated substrates were separated by SDS-PAGE and immunoblotted with the phospho65-Bim, Bim, or GST antibody. Degradation products of bacterially expressed proteins are marked with an asterisk.

Figure 4.

Phosphorylation of Bim_EL at serine 65 promotes Bim_EL-mediated apoptosis. A, Cerebellar granule neurons were transfected with control vector (V), Bim_EL, or Bim_ELS55/65A (55/65A) and β-galactosidase (left panel) or with control vector (V) or JNK DN together with Bim_EL and β-galactosidase (right panel). One day after transfection, cultures were switched from full medium to basal medium in the absence of survival factors. Cells were fixed after 8 hr and subjected to indirect immunofluorescence. Percentage of apoptosis is represented as mean ± SE. The expression of Bim_ELS55/65A induced significantly less apoptosis than Bim_EL (n = 3; ANOVA; p < 0.0005). Expression of JNK DN significantly reduced Bim_EL-induced apoptosis (n = 3; ANOVA; p < 0.005). B, Cerebellar granule neurons were transfected with plasmids encoding nonspliceable cDNAs of Bim_EL, Bim_ELS55A (55A), Bim_ELS65A (65A), Bim_ELS65D (65D), or control vector (V) and β-galactosidase. Cultures were treated as described above and analyzed by indirect immunofluorescence. The expression of Bim_ELS65A induced significantly less apoptosis than Bim_EL and Bim_ELS65D (mean ± SE; n = 4; ANOVA; p < 0.01 and p < 0.0005). The expression of Bim_ELS55A did not reduce the apoptotic effect compared with wild-type Bim_EL (mean ± SE; n = 6; ANOVA; p = 0.4). C, Cerebellar granule neurons were transfected with plasmids encoding nonspliceable cDNAs of wild-type Bim_EL, Bim_ELS65D (65D), or control vector (V) and β-galactosidase. One day after transfection, cultures were switched from full medium to basal medium in the presence (Ins) or absence (-) of insulin (10 μg/ml) to activate the IGF-I receptor. Cells were fixed after 8 hr and subjected to indirect immunofluorescence. Activation of the IGF-I receptor reduced Bim-mediated apoptosis (mean ± SE; n = 4; ANOVA; p < 0.0001) but had little effect on Bim_ELS65D-induced apoptosis (p > 0.04).

Figure 1.

JNK phosphorylates Bim_EL in vitro. A, The structure of Bim_EL is shown schematically. The location of the six putative proline-directed phosphorylation sites, the dynein binding motif (DBM), the BH3-only domain, and the hydrophobic domain (ϕ) are indicated. B, Recombinant GST-Bim_EL was incubated with recombinant JNK1α1 and subjected to an in vitro kinase assay using [γ-³²P]ATP. Phosphorylated GST-Bim_EL was separated by SDS-PAGE, stained with Coomassie blue (bottom panel), and analyzed by autoradiography (top panel). Degradation products of bacterially expressed GST-Bim_EL are indicated with an asterisk. C, Representative tandem mass spectrum of one of the phosphopeptides (amino acids 51-71) derived from JNK-phosphorylated GST-Bim_EL. Fragment ions in the spectrum were sequenced from both the N and C termini (b- and y-type ions). Both serine 55 and serine 65 show additional mass from a phosphate residue.

Figure 3.

JNK phosphorylates Bim_EL in vivo. A, HEK293T cells were transfected with 1 μg of wild-type Bim_EL (WT) or the Bim_EL phosphorylation mutants Bim_ELS55A (55A), Bim_ELS65A (65A), Bim_ELS73A (73A), Bim_ELS100A (100A), and Bim_ELT112A (112A) alone or together with 0.5 μg of activated MEKK1. The electrophoretic mobility of the Bim_EL proteins was assessed by SDS-PAGE and immunoblotting with the HA antibody. B, HEK293T cells were transfected with 1 μg of wild-type Bim_EL or vector control alone or together with 0.5 μg of activated MEKK1, p38MAPK plus MKK3, or activated MEK1. Lysates of transfected cells were immunoblotted with the phospho65-Bim, Bim, phospho-JNK (P-JNK), or phospho-p38MAPK (P-p38MAPK) antibody. Untagged Bim_EL is indicated with an asterisk. C, HEK293T cells were treated with sorbitol (500 mm) for 3 hr. Lysates of cells were immunoblotted with the phospho65-Bim, Bim, phospho-JNK (P-JNK), or phospho-p38MAPK (P-p38MAPK) antibody. D, HEK293T cells were treated with sorbitol (500 mm) for 3 hr in the presence of vehicle or the p38MAPK and JNK inhibitors SB203580 (5 μm) and SP600125 (10 μm), respectively. Lysates of cells were immunoblotted with the phospho65-Bim, Bim, phospho-c-Jun (P-c-Jun), phospho-JNK (P-JNK), JNK, phospho-p38MAPK (P-p38MAPK), or 14-3-3 antibody. Aspecific bands recognized by the phospho65-Bim antibody are indicated with an asterisk.

Figure 5.

p75^NTR-induced apoptosis activates the JNK-dependent phosphorylation of Bim_EL and is dependent on Bim_EL. A, PC12 cells were infected with p75^NTR or control adenovirus (LacZ) together with either 5 or 15 MOI of JBD-JIP (J) or control (L) adenovirus. Lysates were immunoblotted with phospho65-Bim or Bim antibody. Aspecific bands recognized by the phospho65-Bim antibody are indicated with an asterisk. B, Lysates of PC12 cells transfected with increasing amounts of control vector (U6), Bim_EL RNA interference plasmid (Bim RNAi), or control RNAi plasmid (Cdk2 RNAi) were immunoblotted with the Bim (top panel), p42 MAP kinase (ERK1/2; middle panel), or actin antibody (bottom panel). C, PC12rtta cells were transfected with the indicated plasmids and subsequently infected with p75^NTR (p75) or control adenovirus (LacZ). Twenty-four hours after infection, cells were fixed and immunostained for cleaved caspase-3. Double asterisk indicates a difference of p < 0.001 between p75^NTR-infected, pCDNA3-transfected cells (bar 8) and mock or LacZ-infected cells (bars 1-7), and p75^NTR-infected, U6-transfected cells (bar 11) and Bim RNAi-transfected cells (bar 12). Single asterisk indicates a difference of p < 0.05 between p75^NTR-infected, pCDNA3-transfected cells (bar 8) and Bim_ELS65A-transfected cells (bar 10), as determined by ANOVA.