Extracellular signal-regulated kinases 1/2 are serum-stimulated "Bim(EL) kinases" that bind to the BH3-only protein Bim(EL) causing its phosphorylation and turnover

Bim, a "BH3-only" protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of Bim(EL), targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim(EL) phosphorylation remained unclear. We now show that Bim(EL) is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of Bim(EL) occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim(EL), but not Bim(S) or Bim(L), in vitro, and mutation of Ser(65) to alanine blocks the phosphorylation of Bim(EL) by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GST-Bim(EL), but not GST-Bim(L) or GST-Bim(S), in vitro. ERK1/2 also binds to full-length Bim(EL) in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim(EL) protein. Our findings provide new insights into the post-translational regulation of Bim(EL) and the role of the ERK1/2 pathway in cell survival signaling.     

Identification of a DEF-type docking domain for extracellular signal-regulated kinases 1/2 that directs phosphorylation and turnover of the BH3-only protein BimEL

The BH3-only protein, Bim, exists as three splice variants (Bim(S), Bim(L), and Bim(EL)) of differing pro-apoptotic potency. Bim(EL), the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim(EL) splice variant that includes the major ERK1/2 phosphorylation site Ser(65). However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim(EL) (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80-97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim(EL) and also abolished ERK1/2-dependent phosphorylation of Bim(EL) in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim(EL) that may account for the distinct biological properties of this splice variant.     

Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim

The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy.     

Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.     

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.     

Bim and the Pro-Survival Bcl-2 Proteins: Opposites Attract,ERK Repels

Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (BimS, BimL and BimEL). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it’s interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of BimEL. ERK1/2-dependent dissociation of BimEL from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.     

Differential regulation of B-raf isoforms by phosphorylation and autoinhibitory mechanisms

The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf(8b) isoform and exon 9b in the B3-Raf(9b) isoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities, respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We show that exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf(8b) was more pronounced. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf(9b) isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but the S429A mutation activated B2-Raf(8b), whereas it inhibited B3-Raf(9b). These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.     

Grb10 interacts with Bim L and inhibits apoptosis

Bim is a proapoptotic member of the Bcl-2 family and is primarily involved in the regulation of the intrinsic apoptotic pathway. However, the detail of regulation of Bim’s proapoptotic activity has not been clarified yet. Using Bim L as bait, we screened a human fetal cDNA library for interacting proteins and identified Grb10 as an interactor. This interaction was verified by co-immunoprecipitation and intracellular co-localization studies. The potential segment of Bim L that binds Grb10 was identified via a yeast mating test. Grb10 interacted with the DBD (dynein binding domain) of Bim and inhibited apoptosis triggered by overexpression of DBD containing Bim isoforms. The putative phosphorylation sites on DBD of Bim play a role for the anti-proapoptotic activity of Grb10. Our results suggest that Grb10 interacts with Bim L and inhibits its proapoptotic activity in a phosphorylation-dependant manner.     

Multisite phosphorylation regulates Bim stability and apoptotic activity

The proapoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multisite phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the antiapoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses.     

Mutational analysis of the cytoplasmic domain of CEACAM1-4L in humanized mammary glands reveals key residues involved in lumen formation: Stimulation by Thr-457 and inhibition by Ser-461

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion, undergoes extensive alternative splicing, resulting in isoforms with 1-4 Ig-like extracellular domains (ECDs) with either long or short cytoplasmic tails. We have previously shown that CEACAM1-4L (4 ECDs with a long cytoplasmic domain) formed glands with lumena in humanized mammary mouse fat pads in NOD/SCID mice. In order to identify the key residues of CEACAM1-4L that play essential roles in lumen formation, we introduced phosphorylation mimic (e.g., Thr-457 or Ser-461 to Asp) or null mutations (Thr-457 or Ser-461 to Ala) into the cytoplasmic domain of CEACAM1-4L and tested them in both the in vivo mouse model and in vitro Matrigel model of mammary morphogenesis. MCF7 cells stably expressing CEACAM1-4L with the single mutation T457D or the double mutant T457D+S461D, but not the null mutants induced central lumen formation in 3D Matrigel and in humanized mammary fat pads. However, the single phosphorylation mimic mutation S461D, but not the null mutation blocked lumen formation in both models, suggesting that S461 has inhibitory function in glandular lumen formation. Compared to our results for the -4S isoform (Chen et al., J. Biol. Chem, 282: 5749-5760, 2008), the T457A null mutation blocks lumen formation for the -4L but not for the -4S isoform. This difference is likely due to the fact that phosphorylation of S459 (absent in the -4L isoform) positively compensates for loss of T457 in the -4S isoform, while S461 (absent in the -4S isoform) negatively regulates lumen formation in the -4L isoform. Thus, phosphorylation of these key residues may exert a fine control over the role of the -4L isoform (compared to the -4S isoform) in lumen formation.     

Regulation of Bim by TCR signals in CD4/CD8 double-positive thymocytes

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3epsilon injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that Bim(EL) is rapidly phosphorylated in thymocytes in response to CD3epsilon cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-x(L) bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-x(L) in immature thymocytes.     

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca²⁺ releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy. ryanodine receptor; smooth muscle; alternative splicing     

Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells

We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 microm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway.