Affinities of Mcl-1 and Bcl-XL for BH3-Only Proteins Define Distinct Survival Functions at the Outer Mitochondrial Membrane of Multiple Myeloma Cells.

Abstract The Bcl-2 family member Mcl-1 is a critical survival factor for MM cells. However, the functions of Mcl-1 that distinguish it from other anti-apoptotic factors remain unclear. We used fluorescence polarization (FP) to measure the affinity of purified Mcl-1 for amphipathic peptides derived from the BH3-domains of Bad, Bid, Bim, Bik and PUMA, and compared these results with data obtained with Bcl-XL. Functional assays were then performed with mitochondria isolated from 8226 MM cells or clones engineered to overexpress Mcl-1 or Bcl-XL. In contrast to Bcl-XL, Mcl-1 binds to BadBH3 and BikBH3 with very low affinity (>1 uM), confirmed by its inability to bind full-length Bad or Bik proteins. Mcl-1 exhibited high affinity for BimBH3 (90 ± 27 nM), and intermediate affinities for Bid and PUMA. Bcl-XL had intermediate affinities for each of the BH3-peptides tested (175–400 nM). Mitochondria from parental 8226 cells were very sensitive to BidBH3 and BimBH3 as assayed by cytochrome c (cyt c) release; BadBH3 and BikBH3 did not directly induce cyt c release. Mitochondria from 8226-Mcl-1 (which overexpress Mcl-1 5-fold) were very resistant to BidBH3 (even at 500 uM), suggesting that sequestration of BH3-peptide by Mcl-1 was not the sole mechanism of apoptosis inhibition. Paradoxically, Mcl-1-overexpressing mitochondria were more sensitive to BimBH3 (despite the higher affinity of Mcl-1 for Bim), and neither BadBH3 nor BikBH3 could potentiate BidBH3- or BimBH3-induced cyt c release from these mitochondria. In contrast, 8226-Bcl-XL mitochondria were more sensitive to BidBH3, were similarly resistant to BimBH3, and were sensitized by BadBH3 and BikBH3. Protein crosslinking of Mcl-1-overexpressing mitochondria after culture with BidBH3 indicated that peptide sequestration could account for resistance at low peptide concentrations (5–30 uM), but not at higher doses (≥100 uM) where a Bid-induced Bak conformational change occurred as confirmed by exposure of a trypsin-sensitive site. Despite this, formation of Bak multimers was inhibited. (Bax was not detected in 8226 mitochondria). BimBH3 (30 uM) induced detectable cyt c release and Bak dimerization in Mcl-1-overexpressing mitochondria, but 500 uM BimBH3 failed to enhance either effect, and most of the cyt c remained in the mitochondrial pellet. Taken together, these data demonstrate that Mcl-1 and Bcl-XL are not equivalent in their anti-apoptotic functions at the OMM of MM cells. Bad and Bik do not bind Mcl-1, and cannot displace other BH3-only peptides from Mcl-1, suggesting that Mcl-1 has evolved to specifically bind apoptosis-inducing BH3-only proteins in a resistant complex. Mcl-1 confers exceptional protection against BidBH3, and experiments are underway to confirm this with tBid protein. We propose a model in which low levels of BH3-peptide can be sequestered by Mcl-1, while at higher concentrations Mcl-1 binding capacity becomes saturated, allowing peptides to bind Bak and induce a conformational change. However pore formation is inhibited, perhaps by direct binding of Mcl-1 to Bak. To induce pore formation, BH3-peptides must dissociate the Mcl-1/Bak complex: BimBH3, which has a high affinity for Mcl-1 can perform this function, while BidBH3 cannot. This model is consistent with our measured affinity of Mcl-1 for BakBH3 as compared with its affinities for BimBH3 or BidBH3. We suggest that Mcl-1 prevents full cyt c release by preventing activation of the permeability transition pore complex.