Constitutive association of the proapoptotic protein Bim with Bcl-2-related proteins on mitochondria in T cells

Abstract

Apoptosis in activated T cells in vivo requires the proapoptotic Bcl-2 family member Bim. We show here that, despite its ability to bind LC8, a component of the microtubule dynein motor complex, most of the Bim in both healthy and apoptotic T cells is associated with mitochondria, not microtubules. In healthy resting T cells Bim is bound to the antiapoptotic proteins Bcl-2 and Bcl-x L. In activated T cells, levels of Bcl-2 fall, and Bim is associated more with Bcl-x L and less with Bcl-2. Our results indicate that, in T cells, Bim function is regulated by interaction with Bcl-2 family members on mitochondria rather than by sequestration to the microtubules. During immune responses, antigen-specific T cells expand in large numbers and then die (1-3). Sometimes this death involves death receptors (4). However, under some circumstances, the death of activated T cells is driven by changes in the activity of Bcl-2-related proteins.Bcl-2-related proteins are classified by the presence or absence in their sequences of BH1-4 domains. The antiapoptotic family members contain all four of these domains. Family members that are thought to be the executioners of cell death, Bax and Bak, contain only BH1-3. Many family members express only BH3 regions. These proteins are also proapoptotic.It is not clear how Bcl-2 family proteins kill activated T cells. One hypothesis suggests that apoptotic stimuli activate BH3-region-only proteins and these proteins in turn activate Bax or Bak, either by direct interaction, or by neutralizing the antiapoptotic Bcl-2-like proteins (5-7). Alternatively, it has been suggested that antiapoptotic proteins such as Bcl-2 may inhibit the activity of death-dealing caspases. In this model, proapoptotic proteins act by binding to Bcl-2, thus interfering with its inhibition of the caspases.Apoptosis of activated T cells requires the BH3-only Bcl-2 family member Bim (8); however, the processes that regulate Bim activity in T cells are unknown. Some cell types express low levels of Bim constitutively and rapidly increase its levels when they die (9, 10). However, healthy T cells contain appreciable levels of Bim that do not change very much when the cell is activated and undergoes Bim-dependent death (8). In other cell types, Bim activity is controlled by its location. Bim binds with high affinity to LC8, a component of the microtubule-associated dynein motor complex (11). Thus, in some cells, when healthy, Bim is sequestered to the microtubular dynein motor complex. In these cells, death-inducing signals cause release of Bim plus LC8 from the dynein motor complex, translocation of the complex to mitochondria, and Bim-induced death of the cell (11).In this study, we tested the idea that Bim might move from microtubules to mitochondria during activation-induced T cell death. Surprisingly, we found that Bim was not bound to microtubules, even in healthy cells, but rather was associated with mitochondria. In both healthy and dying T cells, Bim was already bound to Bcl-2 and Bcl-x L although the ratio of bin...

Peptide-stimulated DO11.10 T cells divide well but accumulate poorly in the absence of TLR agonist treatment

“…Hildeman et al [18] have further shown that pro-apoptotic Bim is required for activated T cells to die during clonal contraction. Although initially thought to be regulated by subcellular localization [34], Bim has recently been shown to reside in the mitochondria whether or not T cell death is induced by growth factor withdrawal [35]. We tested for Bim expression in this study and found that The MFI for Bcl-x was determined after the indicated treatments and normalized to Bcl-x levels found in CD4 + DO11.10 TCR + cells from untreated mice.…”

Frequent loss of expression of the pro-apoptotic protein Bim in renal cell carcinoma: evidence for contribution to apoptosis resistance

“…In these cells, Bim is probably kept inactive by sequestration to the dynein motor complex on microtubules and activated by its release through an unknown mechanism (Puthalakath et al, 1999). In primary T cells, Bim was reported to be already localized to mitochondria in the absence of apoptosis, and a drop in Bcl-2 and Bcl-x L levels might in this case be the trigger to induce apoptosis (Zhu et al, 2004). Because the activation mechanism of Bim is not understood more clearly, it is difficult to say which stimuli might activate Bim.…”

Regulatory phosphorylation of Bim: sorting out the ERK from the JNK

“…For example, most of the Bim is associated with Bcl-2 and Bcl-x L at the mitochondria rather than at the microtubules in both healthy and apoptotic T cells. 42 The core DLC1-binding region maps to the sequence DKSTQTP (51-57 aa in Bim L and 111-117 aa in Bim EL ) found in exon 4 of Bim L and Bim EL . Certain cellular stresses promote the release of Bim L and Bim EL from microtubules, 9 raising the possibility that this is a dynamic process regulated by stress-induced signalling pathways.…”