International Immunology

International Immunology Advance Access originally published online on November 14, 2006
International Immunology 2006 18(12):1749-1757; doi:10.1093/intimm/dxl109

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TLR-dependent Bim phosphorylation in macrophages is mediated by ERK and is connected to proteasomal degradation of the protein

Georg Häcker¹, Kathrin Suttner¹, Hisashi Harada² and Susanne Kirschnek¹

¹ Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Trogerstrasse 30, D-81675 Munich, Germany
² Department of Internal Medicine, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA

Correspondence to: S. Kirschnek; E-mail: susanne.kirschnek{at}lrz.tum.de

	Abstract

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The pro-apoptotic Bcl-2 homology domain 3-only protein Bim has been shown to play an important role in immune cell homeostasis and various forms of apoptosis in the immune system. Bim is expressed in most immune cells, and regulation of Bim activity can occur on both transcriptional and post-translational levels. Toll-like receptor (TLR) stimulation has previously been shown to increase Bim expression and to cause Bim phosphorylation in the absence of apoptosis in mouse macrophages. Here we identify extracellular signal-regulated kinase as the major kinase responsible for TLR-dependent Bim phosphorylation. Three TLR-dependent serine phosphorylation sites, S55, S65 and S100, on mouse Bim were identified, two of them unique to the splice form Bim_EL and one also present on Bim_L. A Bim mutant defective in these three phosphorylation sites showed slightly enhanced pro-apoptotic activity, which might indicate a protective effect of Bim phosphorylation in this system. Phosphorylation did not alter the association of Bim protein with the microtubule cytoskeleton. However, TLR-mediated phosphorylation led to accelerated degradation of Bim via the proteasome. Thus, TLR stimulation of macrophages can regulate Bim levels in opposing ways, namely by transcriptional induction and by phosphorylation-dependent degradation of the protein.

Keywords: apoptosis, Bcl-2 family

	Introduction

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In the immune system, apoptosis is widely used to control cellular homeostasis, for instance, to reduce the size of T cell clones at the end of an adaptive immune response or to maintain a constant number of neutrophil granulocytes. Apoptosis can be induced by the ligation of death receptors such as Fas/APO-1/CD95. However, recent evidence suggests that apoptosis in the immune system is often mediated via the mitochondrial pathway to apoptosis. In this pathway, the release of mitochondrial cytochrome c is a critical signal transduction step (1). Cytochrome c release is orchestrated by the members of the Bcl-2 protein family (2). Within this family, some members act to inhibit apoptosis (such as Bcl-2 and Bcl-XL). Two proteins, Bax and Bak, are the most downstream mediators of cytochrome c release that are known. The subgroups of Bcl-2 homology domain 3 (BH3)-only proteins are the triggers of this process. During mitochondrial apoptosis, one or several BH3-only proteins are activated and in turn cause the activation of Bax/Bak, while high levels of anti-apoptotic Bcl-2 proteins block this activation (3, 4).

Eight BH3-only proteins are known. Studies with gene-deficient mice suggest that the BH3-only protein Bim/Bod has widespread importance in the immune system. Bim was identified as a Bcl-2-binding protein (5, 6). Regulation of Bim expression and activity seems to be a complex process which involves both transcriptional and post-translational processes. Bim is expressed as at least three different isoforms, Bim_EL, Bim_L and Bim_S, generated by alternative splicing from one transcript (5–7). Bim_EL and Bim_L share a dynein light-chain binding site which allows for sequestration of the proteins to the microtubule cytoskeleton while Bim_S, the shortest isoform, lacks this site (8). In most cell types tested, Bim_EL is the most abundantly expressed isoform and the majority of instances where post-translational modifications have been described concerned Bim_EL.

Bim^–/– mice show elevated lymphoid and myeloid cell numbers, perturbed T cell development and higher susceptibility to autoimmune disease, revealing a crucial role for Bim in leukocyte homeostasis. Bim plays an important role in the contraction of the reactive T cell population at the end of acute T cell responses (9, 10). In lymphocytes, cell death following cytokine deprivation is Bim dependent (11), and withdrawal of cytokines leads to transcriptional up-regulation of Bim (11–15). Down-regulation of Bim by the addition of growth factor has been described for murine hematopoietic progenitor cells (16), neuronal cells (17) and osteoclasts (18).

It has been described a number of times that Bim can be phosphorylated in various cell types. Phosphorylation of Bim protein in lymphocytes positively correlated with protection from apoptosis (19). Phosphorylation of Bim in lymphocytes mediated by growth factor stimulation has been described, and it has been suggested that this process leads to Bim degradation in the proteasome (20, 21). It has further been reported that growth factor-dependent phosphorylation on Ser69 (Ser65 in mouse Bim) followed by proteasomal degradation is mediated by the extracellular signal-regulated kinases (ERKs) (21, 22). In addition, Harada et al. (23) identified three serines that are targets of ERK-dependent phosphorylation sites on Bim_EL. Phosphorylation at these residues has been suggested to regulate IL-3-mediated survival of Fl5.12 pre-B cells (23). Similarly, B cell receptor triggering in a cell line has been shown to induce phosphorylation of Bim_EL. The inhibition of the proteasome has been demonstrated to lead to an accumulation of Bim in this experimental set-up (24). Collectively, these data suggest that Bim levels are physiologically regulated by adjusting Bim turnover.

The precise function of Bim phosphorylation is not entirely clear but likely impacts on its degradation and thus its life span. Bim phosphorylation by Jun NH₂-terminal kinase (JNK) in fibroblasts has been described to inhibit its binding to light chain 8 (LC8) and to increase apoptosis (25). Others have found that JNK-mediated phosphorylation of Bim did not alter its subcellular localization in neurons and either had a pro-apoptotic effect (26) or suppressed pro-apoptotic activity (17). Ley et al. (20) suggested that phosphorylation serves to flag Bim_EL for ubiquitination and subsequent degradation via the proteasome.

We have recently described that Bim is phophorylated during Toll-like receptor (TLR) stimulation of macrophages (27). In the present study, we further explore this event and find that TLR-dependent Bim phosphorylation occurs through ERK on three serine residues. The main effect of this phosphorylation is demonstrated to be an acceleration of Bim turnover through the proteasome.

	Methods

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Cell lines and stimulation of cells
RAW264.7 macrophages were cultured in Low-Tox Clicks RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% FCS (PAA Laboratories, Coelbe, Germany) and antibiotics (100 IU ml^–1 penicillin G and 100 IU ml^–1 streptomycin sulfate). Cells were normally grown in non-culture-coated petri dishes and only for experiments seeded into culture-coated plates. RAW cells were stimulated with the TLR ligands LPS (1 µg ml^–1, Sigma–Aldrich, Steinheim, Germany), Pam3Cys (1 µg ml^–1, EMC Microcollection, Tuebingen, Germany) and CpG-ODN 1668 (1 µM, TibMolBiol).

The MEK1/2 inhibitor U0126 (Calbiochem, Darmstadt, Germany) and the phosphatase inhibitor calyculin A (CalA) (Calbiochem) were used at the indicated concentrations. For proteasome inhibition, MG-132 (Calbiochem) was used. The protein synthesis inhibitor cycloheximide (Cx) was obtained from Sigma–Aldrich.

T-REx HeLa and 293 cells stably expressing the Tet repressor (Invitrogen, Carlsbad, CA, USA) were cultured in DMEM supplemented with 10% tetracycline-free FCS (PAA Laboratories), 100 IU ml^–1 penicillin G, 100 IU ml^–1 streptomycin sulfate and 5 µg ml^–1 Blasticidin.

Generation of mouse bone marrow-derived macrophages
Bone marrow-derived macrophages (BMDMs) were generated according to standard protocols. Briefly, mouse bone marrow was harvested by rinsing the femurs and tibiae. Bone marrow cells (2 x 10⁷ per 10 ml) in a non-culture-coated petri dish were cultured in the presence of 10 ng ml^–1 recombinant mouse M-CSF (R&D Systems). On day 3, another 10 ng ml^–1 M-CSF was added. Adherent cells were harvested on day 9 by accutase treatment (PAA Laboratories) and seeded in 12-well cell culture plates (5 x 10⁵ per well).

Plasmids and cell transfection
Wild-type mouse Bim_EL and a mutant where the three serine sites 55, 65 and 105 were changed to alanine (Bim_ELSA) were expressed as hemagglutinin (HA)-tagged fusion proteins from pCDNA3. For inducible gene expression, constructs were cloned into the tetracycline-inducible pcDNA4/TO/myc HisA vector (Invitrogen).

Transient transfection of cells was performed by electroporation using a Gene pulser electroporator (Bio-Rad, München, Germany). Cells (5 x 10⁶) in 400 µl medium containing 25% FCS and 20 µg of DNA were electroporated with 280 V, 960 µF (RAW cells) or 230 V, 960 µF (HeLa cells).

In case of tetracycline-dependent gene expression in T-REx HeLa cells, tetracycline (1 µg ml^–1) was added 24 h after transfection to induce expression of the target gene.

Western blot analysis
Cells were washed once and harvested in extraction buffer (1% Triton X-100, 50 mM 1,4-piperazinediethanesulfonic acid (PIPES), 50 mM HEPES, 2 mM MgCl₂ and 1 mM EDTA) supplemented with 10 mM NaF, 10 mM sodium orthovanadate and complete proteinase inhibitor mixture (Roche, Mannheim, Germany). For analysis of ERK phosphorylation state, ERK lysis buffer (1% Triton X-100, 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerine, 10 mM Na₄P₂O₇, 1 mM EDTA, 20 mM glycerophosphate, 10 mM NaF, 10 mM sodium orthovanadate and proteinase inhibitor mixture) was used. Nuclei and cellular debris were pelleted by centrifugation at 2000 x g for 10 min at 4°C. Aliquots of the supernatants were used for determination of protein concentration (Bio-Rad assay). Supernatants were boiled in Laemmli buffer and equal amounts of protein were resolved on 12.5% acrylamide gels by SDS-PAGE. Proteins were then transferred on nitrocellulose membranes. Membranes were probed with antibodies specific for Bim, ß-actin (Sigma–Aldrich), phospho-ERK, pan-ERK (Cell Signaling, Danvers, MA, USA) and HA-tag (Upstate, Charlottesville, VA, USA). Secondary HRP-conjugated anti-rabbit or anti-mouse IgG antibodies were obtained from Dianova (Hamburg, Germany). Blots were developed using the enhanced chemiluminescence detection system (PerkinElmer, Boston, MA, USA). For treatment with calf intestinal alkaline phosphatase (AP), cells were lysed in extraction buffer without phosphatase inhibitors and then treated with 20 U/50 µl of calf intestinal AP for 2 h at 37°C. Controls were incubated in the same buffer but without phosphatase and in the presence of phosphatase inhibitors.

2-Dimensional gel electrophoresis
Cells (1 x 10⁶) per sample were lysed in 1% Triton X-100 extraction buffer as described above. Supernatants were acetone precipitated overnight at –20°C, then washed three times with 70% ethanol, dried and re-suspended in 135 µl of lysis buffer [8 M urea, 4% (w/v) CHAPS, 40 mM Tris]. For isoelectric focussing (one dimension), samples were supplemented with 1% immobilized pH gradient buffer (Amersham Pharmacia Biotech, Sweden) and 34 mM dithiothreitol (DTT), incubated for 1 h at room temperature, centrifuged at 13 000 r.p.m. in a microfuge and loaded on stripes with pH 4–7 (Amersham Pharmacia Biotech). The second dimension was performed by SDS-PAGE on 12.5% acrylamide gels followed by western blot analysis as described above. Analysis of gels was performed using Decodon Software (Decodon GmbH, Greifswald, Germany).

Subcellular fractionation
Subcellular fractionation was performed essentially as described (8). Briefly, 10 x 10⁶ cells were lysed in 1% Triton X-100 extraction buffer (1% Triton X-100, 50 mM PIPES, 50 mM HEPES, 2 mM MgCl₂ and 1 mM EDTA) supplemented with 1 mM DTT and complete protease inhibitor mixture (Roche). Lysates were treated with taxol (80 µM; Sigma–Aldrich) and apyrase (10 U ml^–1; Sigma–Aldrich) for 1 h at 37°C. The lysate was then loaded onto a 10% sucrose cushion and centrifuged at 40 000 r.p.m. in a SW41TI rotor in a Beckman ultracentrifuge for 16 h at 4°C. Pellets containing the microtubule fraction were dissolved in Laemmli buffer, and supernatants were concentrated by acetone precipitation. Samples were processed by western blotting as described above.

Sucrose gradient centrifugation was performed by loading the taxol-treated extract on top of a 5–20% discontinuous sucrose gradient (fractions of 0/5/10/15/20% sucrose) followed by ultracentrifugation in a SW41TI rotor at 40 000 r.p.m. for 16 h. The pellet fraction was dissolved in Laemmli buffer, and five fractions of the supernatant were obtained and acetone precipitated. Fractions were resolved by SDS-PAGE and subjected to western blotting as described above.

Assays for cell death
T-REx HeLa cells were transfected by electroporation with inducible Bim_EL wild-type (WT) or mutant expression vectors or empty control vector together with inducible CMV-LacZ. Twenty-four hours later, target gene expression was induced by tetracycline treatment. After additional 24 h, cells were fixed and stained for ß-galactosidase activity. Blue cells were viewed under a microscope and scored alive or dead. Apoptosis rate of transfected cells was quantified by counting at least 200 blue cells in total in triplicate wells.

For quantification of apoptosis by nuclear morphology, T-REx 293 or HeLa cells were transfected by electroporation with inducible Bim_EL WT or mutant expression vectors or empty control vector and 8–24 h later target gene expression was induced with 1 µg ml^–1 tetracycline. After 16 h, cells were Hoechst stained and at least 300 cells per sample were counted.

	Results

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Bim is phosphorylated upon TLR stimulation in macrophages via the MEK/ERK pathway
We have recently described that TLR/myeloid differentiation primary response protein 88 signals induce an increase in the expression of Bim_EL and Bim_L in mouse macrophages. At the same time, these signals cause the appearance of a higher molecular weight (MW) band for both Bim isoforms, and this apparent increase can be reversed by treatment of cell lysates with phosphatase. However, this increase in expression and phosphorylation is not sufficient to induce apoptosis as very little apoptosis is seen in RAW264.7 or BMDM upon stimulation with LPS or CpG-DNA [unless nuclear factor-kappa B (NF-B) is inhibited at the same time] (27).

Stimulation of RAW cells with LPS caused the appearance of a diffuse band of Bim_EL at higher MW while one additional distinct band appeared for Bim_L. Upon treatment of cell lysates with AP, the additional bands were reduced in intensity or disappeared indicating that the described changes were due to phosphorylation of the Bim protein (Fig. 1A).

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Phosphorylation of Bim upon TLR stimulation in macrophages. (A) RAW cells (5 x 105 per well in a 12-well plate) were either left untreated or stimulated with LPS (1 µg ml–1) for 1 h. Extracts were then divided into two parts and incubated with or without AP for 3 h at 37°C. Samples were lysed in 1% Triton X-100 extraction buffer, separated by SDS-PAGE and subjected to western blotting against Bim. (B) One percent Triton X-100 extracts of unstimulated or LPS-stimulated (as above) RAW cells were divided into two fractions and incubated either with or without AP (3 h, 37°C). Samples were subjected to 2D electrophoresis followed by western blotting against Bim. Gels were analyzed by Decodon software, Decodon GmbH, enabling the overlay and comparison of single protein spots between different gels. The overlay shows the two gels to be compared in orange and blue color, respectively. (C) RAW cells (5 x 105 per well in a 12-well plate) were treated with the phosphatase inhibitor CalA for 30 min, and then cells were washed and further incubated for the indicated time periods. Samples were extracted in 1% Triton X-100 extraction buffer and subjected to SDS-PAGE followed by western blotting anti-Bim. (D) RAW cells were seeded as above and were pre-treated with the MEK1/2 inhibitor U0126 (10 µM) for 30 min or not and then stimulated with the TLR ligands LPS (1 µg ml–1), bacterial lipopeptide (Pam3Cys, 1 µg ml–1) or CpG-ODN (1 µM) for 1 h. Samples were extracted in ERK lysis buffer, separated by SDS-PAGE and then subjected to western blotting with anti-Bim. The membrane was re-probed with antibodies against phospho-ERK, total ERK and ß-actin. (E) Primary BMDMs (5 x 105 per well in a 12-well plate) were pre-treated with U0126 or not and then stimulated with LPS (1 µg ml–1) for 1 h. Samples were extracted and subjected to western blotting as in (D).

 
To allow for a better resolution of phosphorylated proteins, 2-dimensional (2D) electrophoresis was performed. Separation of cell extracts from unstimulated RAW cells by 2D electrophoresis followed by anti-Bim western blotting yielded a series of at least seven spots for Bim_EL and four spots for Bim_L. In lysates from LPS-stimulated cells, a shift of the protein spots to more acidic forms was observed (Fig. 1B). AP treatment of cell extracts prior to electrophoresis reversed the number of spots for Bim_EL to five and Bim_L to three, confirming the involvement of phosphorylation. In all cases a number of spots were detected. This may mean that either the AP digest was not complete or that post-translational modifications other than phosphorylation occur in Bim.

To determine the kinase responsible for Bim phosphorylation, a variety of kinase inhibitors was used. Inhibition of the mitogen-activated protein kinases (MAPKs), JNK or p38, PI3 kinase or Src kinase by pharmacological agents showed no detectable effect on Bim phosphorylation (data not shown). However, in the presence of the MEK1/2 inhibitor U0126, the mobility shift of Bim was completely abrogated, correlating with a complete loss of ERK1/2 phosphorylation (Fig. 1D). When macrophages were stimulated with other TLR ligands like bacterial lipopeptide (TLR2) or CpG-DNA (TLR9), inhibition of ERK by U0126 showed the same effect on Bim phosphorylation as seen when LPS as a TLR ligand was used (Fig. 1D). Primary BMDMs showed the same complete abrogation of Bim phosphorylation when ERK was inhibited during TLR stimulation (Fig. 1E). These data identify ERK as the major kinase involved in TLR-induced Bim phosphorylation.

The net phosphorylation of a protein is the result of the balance between phosphorylation and dephosphorylation. We next tested whether phosphatase inhibition by a broad-spectrum inhibitor would also induce Bim phosphorylation. Treatment with CalA for 30 min led to a strong increase in Bim_EL and Bim_L phosphorylation in otherwise unstimulated cells. When CalA was washed away, Bim phosphorylation decreased and returned to normal levels within 8 h. These data suggest that in resting cells Bim is constantly dephosphorylated by constitutively active phosphatases (Fig. 1C).

Identification of three distinct TLR-dependent serine phosphorylation sites on Bim
The appearance of the phosphorylation bands during LPS signaling suggested the involvement of several phosphorylation sites on Bim_EL and one site on Bim_L.

In lymphocytes, distinct serine phosphorylation sites have been identified previously. In a pre-B cell line, IL-3 stimulation led to phosphorylation of three serine residues (23). To test whether these sites were also targets of LPS-activated ERK in macrophages, an HA-tagged Bim_EL mutant (termed HA-Bim_ELSA) was used where serines 55, 65 (unique for Bim_EL) and 100 (present on Bim_EL as well as Bim_L) had been mutated to alanine (23).

HA-Bim_EL or HA-Bim_ELSA was transiently transfected into RAW cells. Two HA-reactive bands were seen in transfected cells at the size expected for Bim_EL and Bim_L, respectively. Since Bim_L is spliced off the Bim_EL transcript (7), this was to be expected.

In unstimulated cells, some HA-Bim_EL protein was found at higher MW, perhaps indicative of RAW cell activation by transfection or by plasmid DNA (Fig. 2A). LPS stimulation of cells transfected with the HA-Bim_EL showed a similar shift in MW as the endogenous protein. In contrast, HA-Bim_ELSA did not show any shift in response to LPS treatment (Fig. 2A). Upon LPS stimulation, a single higher MW band appeared in HA-Bim_L as seen for endogenous Bim_L (Figs 1A and 2A), and this did also not occur in HA-Bim_ELSA transfected cells (Fig. 2A).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Identification of three TLR-dependent Bim phosphorylation sites. (A) RAW cells were transfected with HA-BimEL WT or the mutant, seeded in a 12-well plate (5 x 105 per well) and either left unstimulated or stimulated with LPS (1 µg ml–1) for 1 h. Samples were lysed in 1% Triton X-100 extraction buffer and then subjected to SDS-PAGE followed by western blotting with anti-HA to detect transfected Bim. (B) RAW cells were transiently transfected with HA-BimEL WT or mutant and stimulated for 1 h with LPS (1 µg ml–1). One percent Triton X-100 cell extracts were separated by 2D gel electrophoresis and subjected to western blotting with anti-HA antibody. Analysis of the gels was done with Decodon software (Decodon GmbH) to overlay and compare the single spots. The overlay shows the two gels to be compared in orange and blue color, respectively. (C) RAW cells transfected with HA-BimELWT or HA-BimELSA as described above were stimulated with various concentrations of CalA for 15 min. One percent Triton X-100 extracts were analyzed by SDS-PAGE and western blotting was performed with anti-HA. ß-Actin served as a loading control.

 
When analyzed by 2D electrophoresis, HA-tagged WT Bim showed a clear shift to more acidic forms upon stimulation with LPS while the mutant protein remained unchanged in its migration (Fig. 2B), confirming that the sites mutated in Bim_ELSA were the targets of LPS-dependent phosphorylation of Bim.

We next analyzed the effect of phosphatase inhibition on phosphorylation of HA-Bim_EL and HA-Bim_ELSA. Because it was difficult to get sufficient transfection rates in RAW cells for this experiment, possibly because over-expression of Bim is toxic to cells, we used a tetracycline-inducible expression system in the human epitheloid cell line HeLa. Cells were transfected, induced with tetracycline to express HA-Bim_EL or HA-Bim_ELSA and treated with the phosphatase inhibitor CalA. With increasing inhibitor concentrations, phosphorylation of the HA-Bim_EL protein strongly increased. At high concentrations of CalA, a slight mobility shift of HA-Bim_ELSA was detectable, suggesting the existence of additional phosphorylation sites not used during TLR signaling (Fig. 2C).

Comparative pro-apoptotic activity of HA-Bim_El and HA-Bim_ELSA
These data establish that TLR-dependent ERK stimulation causes the serine phosphorylation of Bim on three residues (Bim_EL) or one residue (Bim_L). Further experiments were directed at understanding the role of this phosphorylation. First, we tested whether HA-Bim_EL and HA-Bim_ELSA differed in their pro-apoptotic activity. HeLa cells were transiently transfected with the two tetracycline-inducible constructs together with the transfection marker ß-galactosidase. The next day, expression was induced by tetracycline for 24 h. Cells were fixed, stained for ß-galactosidase expression and positive cells were scored alive or dead by morphological criteria (28). The pro-apoptotic activity of both proteins was in a similar range although the phosphorylation mutant appeared to be slightly more active (Fig. 3A). In additional experiments, HeLa and 293 cells were transfected as above, and expression of Bim was induced with tetracycline for 16 h. Apoptosis was then quantified by Hoechst staining (Fig. 3B) or measurement of caspase 3 activity (data not shown). The slightly enhanced pro-apoptotic activity of the phosphorylation mutant was confirmed by these experiments.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Comparative pro-apoptotic activity of HA-BimELWT and HA-BimELSA. (A) T-REx HeLa cells were co-transfected with the reporter plasmid pcDNATM4/TO/lacZ and the HA-BimEL expression constructs. Protein expression was induced 24 h after transfection by addition of tetracycline (1 µg ml–1). After 24 h, cells were ß-galactosidase stained and at least 200 cells per sample were judged as live or dead according to their morphology. Values represent mean/SD of triplicate samples. Shown are two independent experiments. (B) T-REx HeLa cells or T-REx 293 cells were transfected with the HA-BimEL expression constructs. Protein expression was induced by addition of tetracycline (1 µg ml–1). Apoptosis was quantified by Hoechst staining after 16 h. At least 300 cells per sample were counted. Shown are two independent experiments for each cell line (black and white bars are separate experiments).

 
Bim phosphorylation does not affect its attachment to the cytoskeleton
Bim_EL and Bim_L have been found to be attached to the dynein motor complex of the microtubule cytoskeleton via their LC8-binding site (8). It is thought that this sequesters Bim and keeps it inactive (8). In an epithelial cell line, it was shown that Bim is released during apoptosis, which was followed by its translocation to the mitochondria.

We tested the possibility that TLR-dependent phosphorylation of Bim caused its release from the cytoskeleton, which might affect its pro-apoptotic activity. RAW cells were transfected with HA-Bim_EL or HA-Bim_ELSA and aliquots of the cells were stimulated with LPS. As before, LPS induced phosphorylation in HA-Bim_EL. However, both phosphorylated and non-phosphorylated forms were found in the fraction containing the microtubule cytoskeleton (Fig. 4). An HA-tagged protein that probably was Bim_S judging by size appeared in some samples. As predicted for Bim_S (which lacks the cytoskeleton attachment site), this isoform was found in the supernatant fraction. There was a tendency towards the appearance of HA-Bim_ELSA in the supernatant fraction (i.e. unbound to the cytoskeleton, Fig. 4A). Although this was unexpected, this may be the explanation for the slightly greater pro-apoptotic potential of HA-Bim_ELSA. Fractionation over a discontinuous sucrose gradient was additionally performed with extracts of 293 cells transfected with either WT or mutant Bim_EL. This resulted in localization of most of Bim_EL in the microtubule-containing pellet, while constitutively mitochondrially localized proteins like Hsp60 or the Bcl-2 family protein Bak appeared in the supernatant fractions (Fig. 4B). No difference between the localization of WT and mutant Bim could be observed in these experiments.

View larger version (63K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Bim phosphorylation does not affect its attachment to the cytoskeleton. (A) RAW cells were transiently transfected with HA-BimELWT or HA-BimELSA and stimulated with LPS (1 µg ml–1) overnight. One percent Triton X-100 cell extracts were prepared and loaded on a 10% sucrose cushion. The microtubule fraction was pelleted by ultracentrifugation (P) and the non-sedimented fraction was acetone precipitated (SN). Samples were separated by SDS-PAGE and analyzed by western blotting with anti-HA. Membranes were re-probed for tubulin as a marker for the microtubule fraction and cytochrome c as a marker for the supernatant fraction. Data are representative of three independent experiments. (B) T-REx 293 cells were transiently transfected with HA-BimELWT or HA-BimELSA. Protein expression was induced with 1 µg ml–1 tetracycline in the presence of z-VAD-fmk. One percent Triton X-100 cell extracts were prepared and loaded on a discontinuous sucrose gradient. The microtubule fraction was pelleted by ultracentrifugation. The pellet fraction P and fractions of decreasing sucrose content were obtained, acetone precipitated and analyzed by western blotting. Samples were separated by SDS-PAGE and analyzed by western blotting with anti-HA. Membranes were re-probed for tubulin as a marker for the microtubule fraction and for Bak and Hsp60 as mitochondrial markers.

 
Bim phosphorylation increases its proteasomal degradation
In other experimental situations, Bim phosphorylation has been shown to increase its turnover due to increased proteasomal degradation. To test this possibility during TLR-induced phosphorylation in macrophages, cells were stimulated with LPS, and the amount of Bim was monitored over time in the presence or absence of the proteasome inhibitor MG-132. As shown in Fig. 5(A), LPS stimulation induced a sustained Bim phosphorylation over 9 h. Additional MG-132 treatment led to discernible accumulation of Bim protein (compare Bim expression in top and bottom panels, Fig. 5A). Surprisingly, the unphosphorylated form of Bim increased more strongly than the phosphorylated form in both Bim_L and Bim_EL. The most straightforward model would be that Bim is phosphorylated and the phosphorylated form is then degraded by the proteasome. However, this model would predict that phosphorylated Bim accumulates when the proteasome is blocked. This finding was therefore unexpected.

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Bim phosphorylation increases its proteasomal degradation. (A) RAW cells were treated with LPS (1 µg ml–1) and MG-132 (1 µM) for the indicated time periods. One percent Triton X-100 extracts were separated by SDS-PAGE and Bim expression was analyzed by western blotting. (B) RAW cells were stimulated with LPS (1 µg ml–1) followed by Cx treatment (50 µM) for the indicated time periods. Cells were lysed in 1% Triton X-100 extraction buffer, separated by SDS-PAGE and Bim was detected by western blotting. Shown are two independent experiments.

 
TLR stimulation not only induces Bim phosphorylation but also increases Bim de novo synthesis, which may confound the analysis of Bim turnover. We therefore performed similar experiments in the presence of the protein synthesis inhibitor Cx. Cells were LPS treated, and Cx was added after 1 h (Fig. 5B, top) or after 2 h (Fig. 5B, bottom). Five hours later (Fig. 5B, top), levels of Bim were compared between cells that were stimulated with LPS prior to Cx treatment and cells that only had been exposed to Cx for the same period of time. As shown in Fig. 5(B) (top), pre-stimulation with LPS accelerated degradation of Bim_EL. At later time points after Cx treatment (18 h in Fig. 5B), Bim_EL had almost completely disappeared in LPS pre-treated cells, while more Bim_EL was still retained in Cx-only treated cells (bottom panel in Fig. 5B). TLR stimulation thus accelerates the degradation of Bim, possibly via its phosphorylation.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Stimulation of macrophages with TLR ligands caused phosphorylation of Bim_L and Bim_EL. This phosphorylation was mediated by the MEK/ERK pathway and occurred on three serine residues: serine 55 and serine 65 are found only in Bim_EL while serine 100 is present in both Bim_L and Bim_EL. Phosphorylation did not appear directly to alter the subcellular localization and therefore probably also not pro-apoptotic activity of Bim. However, LPS stimulation caused the enhanced degradation of Bim by the proteasome. Since TLR stimulation led to both transcriptional induction and degradation of Bim, it appears that two opposing mechanisms are triggered that allow the fine adjustment of Bim levels in the cell.

As noted above, Bim phosphorylation has been noted before in a number of experimental systems. Two functional consequences of this have been considered, a change in pro-apoptotic activity and a change in protein turnover. It is still not fully clear how Bim is activated, and it is therefore difficult to assess the pro-apoptotic activity. An early report showed that Bim_EL and Bim_L are normally sequestered inactively to the microtubule cytoskeleton, from where they are released upon apoptotic stimulation. Bim was further shown then to translocate to mitochondria and to induce apoptosis (8). However, others have suggested that at least a large part of Bim is localized to mitochondria even in healthy T (29) and B lymphocytes (30).

We found both unphosphorylated and phosphorylated Bim almost completely in the fraction containing microtubules. This suggests that phosphorylation does not change the ability of Bim to bind to microtubules and, if release is the means of Bim activation, that phosphorylation does not enhance Bim activity by inducing its release. Phosphorylation of Bim on Thr56 by JNK has been implicated in a change in its subcellular localization. In a transient transfection system, Bim_L phosphorylation was found to inhibit its binding to LC8, thereby releasing Bim from the microtubules and thus favoring apoptosis by increasing the amount of free Bim (25). However, such a mechanism does not appear to operate during ERK-dependent serine phosphorylation of Bim in macrophages.

Phosphorylation of Bim_EL on serine 69 (on human Bim) by the MEK/ERK pathway has been reported earlier to promote proteasomal degradation of the protein (21, 22). In our study, investigation of the effects of TLR-dependent phosphorylation on Bim expression was complicated by the fact that TLR stimulation induced in parallel a significant increase in Bim expression. Experiments in which transcription was blocked showed that the phosphorylated form of Bim is turned over more rapidly, indicating that the phosphorylated form of Bim was preferentially degraded by the proteasome. Intriguingly, proteasome inhibition caused the accumulation of especially the non-phosphorylated form of Bim. This suggests that the amount of phosphorylated Bim in the cell is somehow limited by a feedback mechanism. Alternatively, proteasome inhibition may lead to an increase of Bim phosphatase activity, which could account for a larger amount of unphosphorylated Bim. Taken together, our data are in agreement with a model where Bim phosphorylation during TLR stimulation facilitates Bim degradation and thereby allows for fine-tuning of the amount of Bim in the cell, which probably is a determinant of apoptosis sensitivity. As discussed above, the regulation of Bim activity is still not clear. Accordingly, it is difficult to be certain about the biological relevance of mechanisms that regulate Bim abundance. However, one possibility to consider is that there are fractions of Bim in a cell that are functionally different, such as complexed by Bcl-2-like proteins or attached to microtubules. De novo synthesis, degradation and phosphorylation of Bim may, in addition to regulating abundance, all be involved in shifting molecules between these (hypothetical) fractions of Bim.

MAPKs are activated during TLR signaling. Both ERK and JNK have been described to phosphorylate Bim in lymphocytes and in neuronal cells. In macrophages, we found that inhibition of MEK completely blocked Bim phosphorylation while JNK inhibitors had no effect. One specific ERK phosphorylation site, serine 69 on the human protein (corresponds to serine 65 on mouse Bim_EL), has been described before (21). The present study confirms this. Beyond that, two additional serine residues, serine 55 and 100 appear to be phosphorylated. Direct binding of active ERK to Bim_EL and phosphorylation of serine 65 in vitro and in vivo has been described (22). Whether ERK also directly binds to and phosphorylates serines 55 and 100 or whether this occurs through another kinase in the ERK pathway is unclear at present.

A broad-spectrum phosphatase inhibitor caused massive Bim phosphorylation in the absence of an added TLR stimulus. This indicates an involvement of constitutively active phosphatases and the occurrence of additional potential phosphorylation sites. Thus, the phosphorylation status of Bim seems to be regulated by a balance between kinases and phosphatases, and both increase in kinase activity and decrease in phosphatase activity might be responsible for a shift to stronger phosphorylation.

ERK has often been described as a pro-survival kinase although it is not known how exactly it achieves this effect. Targeting Bim for degradation by its phosphorylation may be one way how ERK primes a cell for survival. We observed a slight increase in pro-apoptotic activity of Bim_EL upon loss of the three identified TLR-dependent serine phosphorylation sites. This is in accordance with the results reported by Harada et al. (23), Ley et al. (22) and Luciano et al. (21) who described a similar effect upon mutation of one or all ofthese sites in lymphocytes. How this might work on a molecular level is unclear. Our data suggest that the mutant is less tightly sequestered to the cytoskeleton, which may simply be the result of a conformational alteration through the mutation. It is also possible that ERK plays a role in binding and inactivating Bim, and mutation of the ERK binding sites would presumably preclude this binding.

Although the most obvious consequence of Bim phosphorylation is a change in apoptosis sensitivity through regulation of Bim levels, it is possible that Bim phosphorylation also impacts on other biological functions and consequences of TLR signaling. For instance, Bcl-2 over-expression is known to slow down cell cycle progression; Bim function, and perhaps phosphorylation through ERK, may also be linked to this pathway. TLR signaling is multi-facetted and touches on a number of different especially transcriptional events. It may be worth pursuing the question of whether TLR signaling is affected in Bim-deficient cells.

Pathways of cell activation and apoptosis are often interconnected. TLRs have the principal potential to induce apoptosis, but this is seen only when NF-B activation is blocked (31). TLR stimulation induces the expression of Bim (27). This is not sufficient to induce apoptosis but it may function to sensitize the cell for apoptosis induction. Phosphorylation, in this model, is the counterpart to balance the amount of Bim in a macrophage during activation through TLR signals.

	Acknowledgements

 
We thank Christina Hilpert and Juliane Vier for expert technical assistance.

	Abbreviations

 

AP, alkaline phosphatase

BH3, Bcl-2 homology domain 3

BMDM, bone marrow-derived macrophage

CalA, calyculin A

Cx, cycloheximide

DTT, dithiothreitol

2D, 2-dimensional

ERK, extracellular signal-regulated kinase

HA, hemagglutinin

JNK, Jun NH2-terminal kinase

LC8, light chain 8

MAPK, mitogen-activated protein kinase

MW, molecular weight

NF-B, nuclear factor-kappa B

PIPES, 1,4-piperazinediethanesulfonic acid

TLR, Toll-like receptor

WT, wild type

	Notes

 
Transmitting editor: D. Wallach

Received 23 May 2006, accepted 19 September 2006.

	References

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