Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4⁺ and CD8⁺ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4⁺ and CD8⁺ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae

The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native α‐tubulin (TUB1) gene, the FP‐Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome‐integrated FP‐tubulin fusions negatively affect microtubule functions. Thus, a simple, economical and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP‐Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of 30 FP‐Tub1 plasmids that use various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function.      

Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.     

Hydrophile scanning as a complement to alanine scanning for exploring and manipulating protein-protein recognition: application to the Bim BH3 domain

Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein-protein and protein-peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild-type residue to alanine removes most of the side-chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex. Hydrophile scanning involves systematic mutation of wild-type residues to a cationic or anionic residue (lysine or glutamic acid, in this case). We find that the results of these mutations provide insights on interactions between polypeptide surfaces that are complementary to the information obtained via alanine scanning. We have applied this technique to a peptide that corresponds to the BH3 domain of the pro-apoptotic protein Bim. The wild-type Bim BH3 domain binds strongly to the anti-apoptotic proteins Bcl-x(L) and Mcl-1. Combining information from the alanine, lysine, and glutamic acid scans has enabled us to identify Bim BH3 domain mutants containing only two or three sequence changes that bind very selectively either to Bcl-x(L) or Mcl-1. Our findings suggest that hydrophile scanning may prove to be a broadly useful tool for revealing sources of protein-protein recognition and for engineering selectivity into natural sequences.     

Maneb potentiates paraquat neurotoxicity by inducing key Bcl-2 family members

An important feature of Parkinson's disease is the degeneration of dopaminergic neurons in the Substantia Nigra pars compacta . Paraquat (PQ) and MPTP cause the selective degeneration of these neurons in vivo , and combining PQ with maneb exacerbates that pathology. Elucidation of the cell death mechanisms involved is important to understand how multiple environmental toxins may contribute to sporadic Parkinson's disease. We recently reported that PQ induces neuronal apoptosis through Bak activation, in contrast to MPP⁺, the toxic metabolite of MPTP, which relies on Bax and p53. Here we show that individually PQ and maneb activate Bak, but together they trigger Bax-dependent cell death. Focusing on mechanisms responsible for this synergy, we found that maneb+PQ increased the expression of three strong Bak inhibitors, Bfl-1, Bcl-xL and Mcl-1, and also induced Bax activators that included Bik and Bim. Those responses favor Bax-dependent MOMP and apoptosis. SiRNA knockdown of Bax and Bak confirmed that individually PQ and maneb induce Bak-dependent cell death, but together they block the Bak pathway and activate apoptosis through Bax.     

Src tyrosine kinase inhibits apoptosis through the Erk1/2- dependent degradation of the death accelerator Bik

Src, the canonical member of the non-receptor family of tyrosine kinases, is deregulated in numerous cancers, including colon and breast cancers. In addition to its effects on cell proliferation and motility, Src is often considered as an inhibitor of apoptosis, although this remains controversial. Thus, whether the ability of Src to generate malignancies relies on an intrinsic aptitude to inhibit apoptosis or requires preexistent resistance to apoptosis remains somewhat elusive. Here, using mouse fibroblasts transformed with v-Src as a model, we show that the observed Src-dependent resistance to cell death relies on Src ability to inhibit the mitochondrial pathway of apoptosis by specifically increasing the degradation rate of the BH3-only protein Bik. This effect relies on the activation of the Ras-Raf-Mek1/2-Erk1/2 pathway, and on the phosphorylation of Bik on Thr124, driving Bik ubiquitylation on Lys33 and subsequent degradation by the proteasome. Importantly, in a set of human cancer cells with Src-, Kras- or BRAF-dependent activation of Erk1/2, resistances to staurosporine or thapsigargin were also shown to depend on Bik degradation rate via a similar mechanism. These results suggest that Bik could be a rate-limiting factor for apoptosis induction of tumor cells exhibiting deregulated Erk1/2 signaling, which may provide new opportunities for cancer therapies.     

Genetically defining the mechanism of Puma- and Bim-induced apoptosis

Using genetically modified mouse models, we report here that p53 upregulated modulator of apoptosis (Puma) and Bcl-2 interacting mediator of cell death (Bim), two pro-apoptotic members of the B-cell lymphoma protein-2 (Bcl-2) family of proteins, cooperate in causing bone marrow and gastrointestinal tract toxicity in response to chemo and radiation therapy. Deletion of both Puma and Bim provides long-term survival without evidence of increased tumor susceptibility following a lethal challenge of carboplatin and ionizing radiation. Consistent with these in vivo findings, studies of primary mast cells demonstrated that the loss of Puma and Bim confers complete protection from cytokine starvation and DNA damage, similar to that observed for Bax/Bak double knockout cells. Biochemical analyses demonstrated an essential role for either Puma or Bim to activate Bax, thereby leading to mitochondrial outer membrane permeability, cytochrome c release and apoptosis. Treatment of cytokine-deprived cells with ABT-737, a BH3 mimetic, demonstrated that Puma is sufficient to activate Bax even in the absence of all other known direct activators, including Bim, Bid and p53. Collectively, our results identify Puma and Bim as key mediators of DNA damage-induced bone marrow failure and provide mechanistic insight into how BH3-only proteins trigger cell death.     

Alternative splicing of Bim and Erk-mediated Bim(EL) phosphorylation are dispensable for hematopoietic homeostasis in vivo

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).