JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis

Abstract

The c-Jun NH(2)-terminal kinase (JNK) is activated when cells are exposed to environmental stress, including UV radiation. Gene disruption studies demonstrate that JNK is essential for UV-stimulated apoptosis mediated by the mitochondrial pathway by a Bax/Bak-dependent mechanism. Here, we demonstrate that JNK phosphorylates two members of the BH3-only subgroup of Bcl2-related proteins (Bim and Bmf) that are normally sequestered by binding to dynein and myosin V motor complexes. Phosphorylation by JNK causes release from the motor complexes. These proapoptotic BH3-only proteins therefore provide a molecular link between the JNK signal transduction pathway and the Bax/Bak-dependent mitochondrial apoptotic machinery.

Figure 1

The BH3-only proteins Bim and Bmf are substrates of JNK. (A) Bacterially expressed GST, GST-BimL, and GST-Bmf were incubated in vitro with activated JNK and [γ-³²P]ATP. The products of the phosphorylation reaction were examined by SDS/PAGE, staining with Coomassie blue (Right), and autoradiography (Left). (B) The structures of BimL and Bmf are illustrated schematically. The BH3 domain and the DLC binding motif are indicated. (C) Mutational analysis of BimL phosphorylation by JNK. In vitro protein kinase assays (KA) were performed by using recombinant Bim with point mutations at Ser-44, Thr-56, and Ser-58 (replaced with Ala). A double point mutation (Ala-56 Ala-58) and a triple point mutation (Ala-44 Ala-56 Ala-58) were also examined. The products of the phosphorylation reaction were examined by SDS/PAGE, staining with Coomassie blue (Lower), and autoradiography (Upper). The numbers above the autoradiograph of the kinase assay represent relative phosphorylation measured by PhosphorImager analysis.

Figure 2

JNK phosphorylates BimL in vivo. (A) Activated JNK causes BimL and Bmf phosphorylation in vivo. Epitope-tagged BimL or Bmf (0.3 μg) were coexpressed together with 0.05 μg of a Bcl2 expression vector. The effect of coexpression (0.3 μg) of an empty vector or expression vectors for JNK1 or activated JNK1 was examined. Control studies were performed by replacing the sites of Thr and Tyr phosphorylation in activated JNK1 with Ala and Phe, respectively. The electrophoretic mobility of BimL and Bmf was examined by immunoblot (IB) analysis. (B) Epitope-tagged BimL (0.3 μg) was coexpressed together with 0.05 μg of a Bcl2 expression vector in cells labeled with [³²P]phosphate. The BimL was isolated by immunoprecipitation and examined by SDS/PAGE, electrotransfer onto a poly(vinylidene difluoride) membrane, autoradiography (Upper), and immunoblot analysis (Lower). The effect of mutation of the JNK phosphorylation sites and exposure to UV radiation (60 J/m²) were examined. (C) The phosphorylated wild-type and T56A BimL were examined by phosphoamino acid analysis performed by partial acid hydrolysis and thin-layer electrophoresis. The migration of phosphoserine, phosphothreonine, and phosphotyrosine standards is indicated. [³²P]Phosphothreonine was detected in wild-type BimL but was not detected if Thr-56 was replaced with Ala.

Figure 3

Phosphorylated BimL is released from DLC1. (A and B) Cells were cotransfected with 0.3 μg of epitope-tagged BimL and DLC1 expression vectors together with 0.05 μg of a Bcl2 expression vector. The effect of coexpression of activated JNK1 was investigated. The presence of BimL and DLC1 in the cell lysate was examined by immunoblot (IB) analysis. Coimmunoprecipitation analysis was performed by immunoblot analysis of DLC1 immunoprecipitates (IP) with an antibody to Bim. The results of mutational analysis of the Bim phosphorylation sites Ser-44, Thr-56, and Ser-58 replaced with Asp (A) or with Ala (B) are shown. (C) DLC1 interacts with nonphosphorylated BimL. Cells were cotransfected with 0.3 μg of BimL, 0.05 μg of Bcl2, 0.3 μg of activated JNK1, and 0.5 μg of DLC1 expression vectors. The amount of BimL and DLC1 in cell lysates and in DLC1 immunoprecipitates was examined by immunoblot analysis. Phosphorylated BimL with reduced electrophoretic mobility was not coimmunoprecipitated with DLC1.

Figure 4

Mutational analysis of the JNK phosphorylation site Thr-56 on BimL-stimulated apoptosis. (A and B) Cells were cotransfected with 0.4 μg of an empty vector or with an expression vector for BimL. The effect of replacement of the JNK phosphorylation site Thr-56 with Ala and Asp was examined. In addition, the effect of mutational disruption of the BH3 domain (Leu-92 and Ile-95 replaced with Asp and Glu, respectively) was investigated. The cells were harvested 40 h after transfection. Cell viability (A) and DNA fragmentation (B) were measured. The data presented are the mean ± SD (n = 3). (C) Cells were cotransfected with pEGFP (CLONTECH) together with a BimL expression vector. The cells expressing EGFP were examined 16 h after transfection by flow cytometry after staining with phycoerythrin-conjugated annexin V and 7-aminoactinomycin D. Cells of the annexin V-positive population of 7-aminoactinomycin D-negative cells (Upper Left) are apoptotic.

Figure 5

A JNK-dependent apoptosis signaling pathway. (A) Schematic illustration of MAPK phosphorylation of BimL and BimEL. Two groups of MAPK sites were identified. Three Ser-Pro sites are phosphorylated by the ERK group of MAPK within the BimEL-specific insert region. In addition, three JNK phosphorylation sites are located within the common region of BimL and BimEL that is absent from BimS. Phosphorylation of Bim on Thr-56 causes dissociation from DLC1. (B) Schematic illustration of a JNK-dependent apoptosis signaling pathway. This signaling pathway is initiated by the phosphorylation of Bim and Bmf by JNK. In some cells, JNK may also contribute to Bim expression. The phosphorylation by JNK is proposed to cause the release of Bim and Bmf from dynein and myosin V motor complexes. The activated Bim and Bmf may directly activate Bax and Bak or may indirectly activate Bax and Bak by binding antiapoptotic Bcl2 family proteins (e.g., Bcl2 and Bcl-X_l). These mechanisms can lead to the engagement of the Bax/Bak-dependent mitochondrial pathway of apoptosis. Smac, second mitochondria-derived activator of caspase; HtrA2, second homology of the bacterial HtrA endoprotease; AIF, apoptosis-inducing factor; and EndoG, endonuclease G.