Abstract
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-XL 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-XL revealed that the phospho-Ser158 makes favorable interactions with two BCL-XL 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.
Highlights
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]
According to the crystal structure of the mouse BIM BH3 domain bound to BCL-XL, 21 residues of BIM form the core region of the BH3 domain that spans the surface groove of BCL-XL.[14]
It was expected that a BH3BIM variant containing serine in place of Glu[158] would exhibit reduced binding affinity for the antiapoptotic BCL-2 family proteins, but phosphorylation of Ser[158] could restore the binding affinity since phosphorylated serine often serves as a mimic of glutamate residue
Summary
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.[18]. Apoptosis is attenuated in cancer cells because of the abundance of antiapoptotic BCL-2 proteins and/or prevention of apoptosis induction. 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]
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