Conversion of cell-survival activity of Akt into apoptotic death of cancer cells by two mutations on the BIM BH3 domain

Survival and proliferation of cancer cells are often associated with hyperactivity of the serine/threonine kinase, Akt. Herein, we show that prosurvival activity of Akt can be converted into prodeath activity by embedding an Akt recognition sequence in the apoptogenic BH3 domain of human BIM. The recognition sequence was created by introducing two mutations, I155R and E158S, into the core region of the BIM BH3 domain. Although a 21-mer BIM BH3 peptide containing these two mutations bound weakly to BCL-X_L and BCL-2, this peptide with phosphorylation of Ser158 bound to these proteins with a dissociation constant of <10 nM. The crystal structure of the phosphorylated peptide bound to BCL-X_L revealed that the phospho-Ser158 makes favorable interactions with two BCL-X_L residues, which cannot be formed with unphosphorylated Ser158. Remarkably, the designed peptide showed a cytotoxic effect on PTEN-null PC3 tumor cells whose Akt activity is aberrantly high. The cell-killing activity disappeared when the cellular Akt activity was lowered by ectopic PTEN expression. Thus, these results lay a foundation for developing a peptide or protein agent that is dormant in normal cells but is transformed into a potent apoptogenic molecule upon phosphorylation by hyperactivity of Akt in cancer cells.The interplay between the BCL-2 family proteins regulates mitochondrion-mediated apoptotic cell death.^{1, 2} The BCL-2 family proteins are characterized by having at least one BCL-2 homology (BH) domain, and they are classified into three distinct subgroups based on their functional and structural features. One subgroup consists of BAX and BAK, which contain the BH1-BH4 domains and mediate apoptosis by increasing the permeability of the mitochondrial outer membrane (MOM) and thus leading to the release of the apoptogenic factors, such as cytochrome c and Smac/Diablo.^{3, 4, 5, 6} Another subgroup is composed of antiapoptotic proteins, BCL-2, BCL-X_L, BCl-w, MCL-1, A1 and BCL-B, which contain the BH1-BH4 domains that are arranged to form an extended hydrophobic groove known as the BH3-binding groove.⁷ The remaining subgroup is composed of a diverse set of proteins that are unrelated to each other except for the possession of the BH3 domain.⁷ These BH3-only proteins sense and convey apoptotic cell death signals, ultimately leading to the activation of BAX and BAK.^{8, 9} The antiapoptotic BCL-2 subfamily proteins bind the BH3 domain of BAX/BAK and of the BH3-only proteins through their BH3-binding groove.^{10, 11, 12, 13, 14, 15}Biochemical studies have discovered that a number of the BH3-only proteins termed ‘activators'', such as BID and BIM, bind directly to BAX and induce its activation, whereas other BH3-only proteins termed ‘sensitizers'' induce apoptosis by releasing the activators sequestered by the antiapoptotic proteins.^{5, 16, 17} A recent crystallographic study revealed that the BID BH3 peptide binds to the canonical BH3-binding groove of BAX and induces a pronounced conformational change that exposes the BH3 domain of BAX.¹⁸ The activated BAX oligomerizes to induce the permeabilization of the MOM.⁶ The antiapoptotic BCL-2 proteins were suggested to sequester the BH3 domains of both BAX and the activator BH3-only proteins to prevent the BAX oligomerization.¹⁸Apoptosis is attenuated in cancer cells because of the abundance of antiapoptotic BCL-2 proteins and/or prevention of apoptosis induction. Anticancer BH3 peptides have been developed, especially those derived from BIM, which interacts with all of the antiapoptotic proteins with extremely high affinity.^{15, 19} These BH3 peptides exhibit a broad and multimodal targeting of the BCL-2 family proteins.^{20, 21, 22} Promising small molecular anticancer compounds have also been developed that mimic the BH3 peptides and bind to the surface groove of the antiapoptotic proteins.²³ ABT-737 and ABT-263 selectively bind to and lower the amounts of the functional BCL-2, BCL-X_L and BCL-w proteins to induce the apoptotic death of tumor cells that depend especially on the overexpression of the three proteins.^{24, 25} The BH3 peptides and the BH3 mimetics both bear an intrinsic shortcoming in that they inhibit the BCL-2 family proteins not only in cancer cells but also in normal cells as they cannot distinguish cancerous from normal cells.One of the hallmarks of many cancer and tumor cells is the hyperactivation of the serine/threonine (Ser/Thr) protein kinase Akt, which is a key signaling molecule in the cellular survival pathway.²⁶ In many types of cancers, including glioma, prostate cancer and breast cancer, Akt is required to maintain a proliferative state and for progression into a more malignant state in conjunction with genetic mutations.^{26, 27, 28}We set out to develop a molecule that can respond to the hyperactivity of Akt and can lead to the death of cancer cells. Herein, we describe the embedment of the Akt recognition sequence into the BIM BH3 peptide and the cancer cell-specific apoptogenic property of the resulting BIM BH3 peptide variant characterized by X-ray crystallography, calorimetry and cell-based biochemistry.