Mono- or Double-Site Phosphorylation Distinctly Regulates the Proapoptotic Function of Bax

Bax is the major multidomain proapoptotic molecule that is required for apoptosis. It has been reported that phosphorylation of Bax at serine(S) 163 or S184 activates or inactivates its proapoptotic function, respectively. To uncover the mechanism(s) by which phosphorylation regulates the proapoptotic function of Bax, a series of serine (S)→ alanine/glutamate (A/E) Bax mutants, including S163A, S184A, S163E, S184E, S163E/S184A (EA), S163A/S184E (AE), S163A/S184A (AA) and S163E/S184E (EE), were created to abrogate or mimic, respectively, either single or double-site phosphorylation. The compound Bax mutants (i.e. EA and AE) can flesh out the functional contribution of individual phosphorylation site(s). WT and each of these Bax mutants were overexpressed in Bax−/− MEF or lung cancer H157 cells and the proapoptotic activities were compared. Intriguingly, expression of any of Bax mutants containing the mutation S→A at S184 (i.e. S184A, EA or AA) represents more potent proapoptotic activity as compared to WT Bax in association with increased 6A7 epitope conformational change, mitochondrial localization/insertion and prolonged half-life. In contrast, all Bax mutants containing the mutation S→E at S184 (i.e. S184E, AE or EE) have a mobility-shift and fail to insert into mitochondrial membranes with decreased protein stability and less apoptotic activity. Unexpectedly, mutation either S→A or S→E at S163 site does not significantly affect the proapoptotic activity of Bax. These findings indicate that S184 but not S163 is the major phosphorylation site for functional regulation of Bax's activity. Therefore, manipulation of the phosphorylation status of Bax at S184 may represent a novel strategy for cancer treatment

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed

BIF-1 inhibits both mitochondrial and glycolytic ATP production: its downregulation promotes melanoma growth.

Endophilin B1, also known as BAX-interacting protein 1 (BIF-1), is part of the endophilin B protein family, and is a multifunctional protein involved in the regulation of apoptosis, autophagy, and mitochondrial morphology. The role of BIF-1 in cancer is controversial since previous reports indicated to both tumor-promoting and tumor-suppressive roles, perhaps depending on the cancer cell type. In the present study, we report that BIF-1 is significantly downregulated in both primary and metastatic melanomas, and that patients with high levels of BIF-1 expression exhibited a better overall survival. Depleting BIF-1 using CRISPR/Cas9 technology in melanoma cells resulted in higher proliferation rates both in vitro and in vivo, a finding that was associated with increased ATP production, metabolic acidification, and mitochondrial respiration. We also observed mitochondrial hyperpolarization, but no increase in the mitochondrial content of BIF-1-knockout melanoma cells. In contrast, such knockout melanoma cells were equally sensitive to anticancer drug- or UV irradiation-induced cell death, and exhibited similar autophagic activities as compared with control cells. Taken together, it appears that downregulation of BIF-1 contributes to tumorigenesis in cutaneous melanoma by upregulating mitochondrial respiration and metabolism, independent of its effect on apoptosis and autophagy

Shaping membranes into autophagosomes

The class III phosphatidylinositol 3-kinase( Pi( 3) KC3) is part of a key complex regulating autophagy. How this complex mechanistically contributes to autophagosome formation remains largely unknown. The BaR-domain- containing protein Bif-1/ endophilin B1, commonly associated with the control of membrane curvature, has been identified as a new interacting partner for Pi( 3) KC3, suggesting the complex plays an active part in the regulation of membrane dynamics during autophagy