Transcripts derived from the Bim
gene are alternatively spliced to create several different Bim proteins (Bouillet et al., 2001b
). BimEL is encoded by sequences derived from exons 2–6 (). Alternative splicing can delete sequences derived from exon 3 (BimL) or exons 3 & 4 (BimS) to create additional Bim isoforms. These alternatively spliced exons encode the major sites of Bim phosphorylation (). To study the role of Bim phosphorylation, we examined the effect of replacement of these phosphorylation sites with Ala residues. Transfection studies using a cDNA expression vector demonstrated that the mutant Bim proteins can be expressed (). Furthermore, co-immunoprecipitation analysis demonstrated that the mutant proteins were able to interact with the pro-survival Bcl2-family protein Mcl-1 (). Substitution of the major Bim phosphorylation sites with Ala residues therefore does not result in the expression of Bim proteins that completely lack functional activity. These data suggest that the physiological role of Bim phosphorylation can be tested by phenotypic analysis of mutant mice that express phosphorylation-defective Bim proteins.
Creation of mice with defects in Bim phosphorylation
To study the role of Bim phosphorylation, we constructed mice with germ-line point mutations in the Bim gene using homologous recombination in ES cells (). A targeting vector was designed to insert a floxed NeoR cassette within intron 4 and introduce specific mutations in exons 3 and 4. The NeoR cassette was excised with Cre recombinase to create a Bim genomic locus with a single LoxP site within intron 4. We created four mouse strains with this single LoxP site in intron 4. First, we constructed mice that lack mutations within the coding regions of the Bim gene. These mice (BimLoxP) express wild-type Bim protein. Second, we constructed mice with mutations within Bim exon 3 that replace the three MAP kinase phosphorylation sites (Ser-55/65/73) with Ala residues (Bim3SA). Translationally silent nucleotide changes were introduced to create novel restriction sites (NcoI, PstI, and XhoI) at each of the mutated phosphorylation sites. Third, mice were constructed with mutations within exon 4 that replace the JNK phosphorylation site Thr-112 with Ala (BimT112A) and create a novel restriction site (KasI). Fourth, we constructed mice (BimΔEL) with a deletion of alternatively spliced exon 3. Heterozygous matings of the mutant mice led to the creation of homozygous mice for the mutant Bim alleles (). The average litter size obtained from matings of homozygous mice with mutant Bim alleles was not significantly different (p > 0.05) from matings of wild-type mice.
Construction of mice with phosphorylation-defective Bim
We examined Bim protein expression by immunoblot analysis of extracts prepared from the thymus and spleen. The major Bim isoform detected in wild-type mice was BimEL, but smaller amounts of BimL were also found (). The low abundance BimS isoform was not reproducibly detected. A similar pattern of Bim expression was observed in studies of control BimLoxP mice. This finding indicates that the presence of a single LoxP site within intron 4 does not markedly alter Bim expression. A similar expression pattern of Bim proteins was observed in BimT112A mice and Bim3SA mice. In contrast, no BimEL was detected in BimΔELmice. The BimΔEL mice expressed increased amounts of BimL because of the deletion of alternatively spliced exon 3.
Together, these data establish that control BimLoxP mice express normal amounts of wild-type Bim proteins. Moreover, the mutant Bim3SA, BimT112A, and BimΔEL mice that express phosphorylation-defective Bim proteins are viable.
Bim is a target of MAP kinase phosphorylation in vivo
To test whether Bim is subject to multi-site phosphorylation in vivo
, we examined the phospho-isomers of BimEL using 2D gel electrophoresis (Bunin et al., 2005
; Hacker et al., 2006
; Kuroda et al., 2006
; Puthalakath et al., 2007
). Immunoblot analysis demonstrated that BimEL in serum-starved MEF exhibited a heterogeneous distribution between a number of phospho-isoforms (). Serum-treatment caused a large shift in the distribution of the BimEL phospho-isoforms to a more homogeneous group with higher negative charge, consistent with increased phosphorylation on multiple sites. Studies of homozygous Bim3SA
MEF indicated that the replacement of the three major MAP kinase phosphorylation sites (Ser-55/65/73) with Ala strongly suppressed the effect of serum on BimEL phospho-isomers (). In contrast, studies of homozygous BimT112A
MEF demonstrated that the replacement of the Thr-112 phosphorylation site with Ala did not prevent the major effects of serum on BimEL phospho-isomers (). These data are consistent with previous reports that Ser-55/65/73 represent major sites of phosphorylation by serum-stimulated ERK and that Thr-112 is a major site of Bim phosphorylation by stress-activated JNK (Ley et al., 2005
Analysis of Bim phosphorylation in vivo
To examine the phosphorylation of BimEL on specific sites we performed immunoblot analysis using phospho-specific antibodies. Phosphorylation of BimEL on the MAP kinase phosphorylation site Ser-65 can be detected using previously characterized antibodies (Putcha et al., 2003
). However, an antibody to the JNK phosphorylation site Thr-112 has not been described. We therefore prepared a phospho-specific antibody to the Thr-112 phosphorylation site on BimEL. Immunoblot analysis demonstrated that this antibody detected recombinant BimEL in cells expressing activated JNK and that replacement of Thr-112 with Ala prevented the detection of BimEL (). Together, these data indicate that BimEL phosphorylation on Ser-65 and Thr-112 can be examined by immunoblot analysis.
Phosphorylation of Bim on Ser65 and Thr112 in vivo
Treatment of BimLoxP
MEF with serum caused markedly increased phosphorylation of wild-type BimEL on Ser-65 (). A similar amount of serum-induced phosphorylation on Ser-65 was detected in homozygous BimT112A
MEF, but no Ser-65 phosphorylation was detected in homozygous Bim3SA
MEF (). Exposure of the MEF to stress (UV radiation) caused no change in the phosphorylation of these Bim proteins on Ser-65 (). Together, these data are consistent with the conclusion that Ser-65 is phosphorylated by serum-stimulated ERK (Ley et al., 2005
) and that mutation of the Thr-112 phosphorylation site does not affect the phosphorylation of BimEL on Ser-65 ().
Exposure of primary BimLoxP MEF to stress (UV radiation) caused increased phosphorylation of wild-type BimEL on Thr-112 (). A similar amount of UV-induced phosphorylation on Thr-112 was detected in homozygous Bim3SA MEF, but no Thr-112 phosphorylation was detected in homozygous BimT112A MEF (). To test whether this UV-stimulated phosphorylation was mediated by JNK, we examined BimEL phosphorylation in compound mutant Jnk1−/− Jnk2−/− fibroblasts that express no JNK. These studies demonstrated that no UV-stimulated phosphorylation of BimEL on Thr-112 was detected in JNK-deficient fibroblasts (). Together, these data indicate that JNK is the physiogically relevant UV-stimulated kinase that phosphorylates BimEL on Thr-112 () and that mutation of the Ser-65 phosphorylation site does not affect the phosphorylation of BimEL on Thr-112 ().
One unexpected finding was that BimEL was also phosphorylated on Thr-112 in response to serum-stimulation (). It was unlikely that this phosphorylation was solely mediated by JNK because it is established that the stress-regulated JNK pathway is not strongly activated by serum-stimulation (Davis, 2000
). Indeed, studies of JNK-deficient fibroblasts indicated that JNK was not required for serum-stimulated phosphorylation of BimEL on Thr-112 (). A different class of MAP kinase may account for this serum-stimulated phosphorylation. A role for p38 MAP kinase was possible, but UV-stimulated p38 MAP kinase activity in JNK-deficient fibroblasts did not cause BimEL phosphorylation on Thr-112 (). We therefore tested the contribution of ERK to serum-stimulated BimEL phosphorylation on Thr-112. Studies of JNK-deficient fibroblasts demonstrated that serum-stimulated BimEL phosphorylation on Thr-112 was strongly suppressed by an inhibitor (U0126) of the ERK signaling pathway (). Together, these data indicate that Thr-112 is a target of both the ERK and JNK signaling pathways and that Thr-112 phosphorylation is mediated primarily by JNK in response to UV radiation and by ERK in response to serum-stimulation (). In contrast, Ser-65 is a selective target of the ERK pathway in serum-stimulated fibroblasts (Ley et al., 2005
) and Ser-65 is not phosphorylated by JNK in response to the exposure of fibroblasts to UV radiation ().
Interaction of Bim with members of the Bcl2 family
It is established that one mechanism that mediates Bim apoptotic activity is an interaction of Bim with other members of the Bcl2 family, including the anti-apoptotic proteins Bcl2, Bcl-XL, and Mcl-1 (Willis et al., 2007
). We therefore performed co-immunoprecipitation assays to test whether mutation of Bim phosphorylation sites caused altered interaction of Bim with Bcl2 family proteins. Studies of primary MEF prepared from homozygous BimLoxP
, and BimKO
mice demonstrated that the anti-apoptotic proteins Bcl2, Bcl-XL, and Mcl-1 co-immunoprecipitated with Bim (). In contrast, the pro-apoptotic protein Bax did not co-immunoprecipitate with Bim (), consistent with previous observations (Willis et al., 2007
Interaction of Bim with Bcl2 family proteins
Mutation of Bim phosphorylation sites (Bim3SA
) did not cause marked changes in the co-immunoprecipitation of Bim with Mcl-1 or Bcl-XL (). Similarly, both wild-type Bim and Bim3SA
co-immunoprecipitated with Bcl2. In contrast, BimT112A
did not co-immunoprecipitate with Bcl2 (). Since the binding of Bim to Bcl2 contributes to Bim-induced apotptosis (Willis et al., 2007
), these data suggest that the BimT112A
protein may cause reduced apoptosis in cells that depend on Bcl2 for survival.
Phosphorylation sites encoded by alternatively spliced exon 3 contribute to the regulation of Bim expression
Serum-starvation of primary MEF causes increased Bim expression that is associated with decreased activation of ERK MAPK (). In contrast, serum-treatment caused ERK activation and decreased Bim expression (). Previous studies have established that changes in Bim levels are mediated by altered Bim
gene expression and Bim protein stability (Strasser, 2005
). Indeed, the Bim protein is rapidly degraded by a ubiquitin-mediated mechanism in response to ERK MAP kinase activation (Ley et al., 2003
; Luciano et al., 2003
). Thus, the serum-induced decrease in Bim protein expression observed in primary MEF is attenuated if the cells are incubated with the MEK inhibitor U0126 to block ERK MAPK activation or with the proteasome inhibitor MG132 ().
The BimEL specific domain encoded by exon 2 is required for MAPK-induced Bim degradation
To test the role of Bim phosphorylation during serum-induced Bim protein degradation, we compared the time course of Bim degradation in primary MEF prepared from embryos that express wild-type and phosphorylation-defective Bim proteins. Control studies using MEF obtained from BimLoxP
mice demonstrated that serum caused a rapid reduction in the electrophoretic mobility of BimEL prior to degradation (). Similar serum-induced degradation of BimEL protein was detected in MEF isolated from homozygous BimT112A
embryos (). In contrast, no serum-induced loss of BimEL protein expression was detected in MEF prepared from homozygous Bim3SA
mice (). These data indicate that the MAP kinase phosphorylation sites encoded within exon 3 of the Bim
gene are required for serum-induced Bim degradation. To test this hypothesis, we examined MEF isolated from BimEL
mice that lack alternatively spliced exon 3 which encodes the three sites of MAPK phosphorylation (Ser-55/65/73). These studies demonstrated that the deletion of exon 3 results in the expression of BimL as the major Bim isoform () and that this Bim protein is resistant to serum-induced degradation ().
To examine the functional significance of phosphorylation-induced changes in Bim protein expression, we examined the apoptotic responses of primary MEF. Previous studies have demonstrated that MEF isolated from Bim knockout (BimKO
) mice retain sensitivity to many stresses, indicating that Bim may play only a redundant role in stress-induced apoptosis of MEF (Naik et al., 2007
). Nevertheless, BimKO
MEF are resistant to serum withdrawal-induced apoptosis (Ewings et al., 2007
). We therefore tested the serum withdrawal-induced apoptotic response of primary MEF isolated from mice that express either wild-type or phosphorylation-deficient Bim proteins.
MEF that express wild-type Bim (BimLoxP) exhibited apoptotic fragmentation of genomic DNA in response to serum withdrawal (). This apoptotic response was suppressed in Bim knockout MEF (BimKO). Studies of MEF isolated from BimT112A mice indicated that the Thr-112 phosphorylation site did not contribute to the apoptotic response (). In contrast, loss of the ERK MAP kinase phosphorylation sites encoded by exon 3, caused by substitution of Ser-55/65/73 with Ala residues (Bim3SA) or deletion of exon 3 (BimΔEL), increased serum withdrawal-induced apoptosis (). This increased apoptotic response is consistent with the hypothesis that the MAP kinase phosphorylation sites encoded by exon 3 act to inhibit Bim function. These phosphorylation sites may reduce Bim apoptotic activity by causing proteasomal degradation of Bim ().
A phosphorylation site encoded by alternatively spliced exon 4 contributes to the regulation of Bim apoptotic activity
Studies of Bim
) mice indicate that Bim plays a major role in the apoptotic response of thymocytes and T cells (Strasser, 2005
). Indeed, BimKO
mice exhibit defects in the deletion of autoreactive thymocytes and the response to stress (Bouillet et al., 1999
; Bouillet et al., 2002
). To test whether Bim phosphorylation may contribute to these Bim-dependent apoptotic processes, we examined thymocyte apoptosis in mutant mice that express phosphorylation-defective Bim proteins.
Treatment of wild-type mice with the anti-inflammatory drug dexamethasone caused apoptosis of double-positive CD4+
(DP) thymocytes. This deletion of DP thymocytes is partially suppressed in BimKO
mice (Erlacher et al., 2005
). Studies of control BimLoxP
mice and mice with phosphorylation-defective Bim
, and BimΔEL
) by flow cytometry demonstrated comparable sub-populations of CD4/8 thymocytes. Treatment with dexamethasone caused similar deletion of DP thymocytes in BimLoxP
, and BimΔEL
mice (). This observation suggests that the MAP kinase phosphorylation sites encoded by exon 3 do not contribute to dexamethasone-stimulated apoptosis of DP thymocytes. In contrast, reduced deletion of DP thymocytes was observed in BimT112A
mice following treatment with dexamethasone (). These data suggest that the phosphorylation site Thr-112 may contribute to the regulation DP thymocyte apoptosis.
Effect of phosphorylation-defective Bim on thymocyte apoptosis in vivo
mice exhibit defects in the deletion of autoreactive thymocytes (Bouillet et al., 2002
). To test the role of Bim phosphorylation, we initially examined the effect of engagement of the T cell receptor with anti-CD3 to cause deletion of DP thymocytes. These studies demonstrated that the administration of anti-CD3 caused similar deletion of DP thymocytes in BimLoxP
, and BimΔEL
mice, but BimT112A
mice exhibited reduced deletion of DP thymocytes. These data suggested that the Thr-112 phosphorylation site may contribute to negative selection of thymocytes. To test this hypothesis we examined the deletion of autoreactive thymocytes in a mouse model with transgenic expression of a T cell receptor (TCR) that recognizes the HY male-specific antigen. In this model, male (but not female) mice exhibit strong negative selection of thymocytes (Kisielow et al., 1988
We found that female HY-TCR+ and HY-TCR− mice exhibited similar CD4/8 thymocyte sub-population profiles in the context of homozygous control (BimLoxP) and phosphorylation-defective Bim alleles (Bim3SA, BimΔELand BimT112A). In contrast, studies of male HY-TCR+ mice demonstrated marked deletion of autoreactive DP thymocytes compared with male HY-TCR− mice. The extent of deletion of autoreactive thymocytes was similar in studies of male HY-TCR+ BimLoxP, Bim3SA, and BimΔELmice (). However, reduced deletion of DP thymocytes was observed in male HY-TCR+BimT112A mice (). These data indicate that mutation of the phosphorylation site Thr-112 causes reduced apoptosis of autoreactive DP thymocytes. This reduction in apoptosis correlates with the observation that the phosphorylation-defective BimT112A protein exhibits a defect in binding Bcl2 ().