Cytotoxic amphiphiles and phosphoinositides bind to two discrete sites on the Akt1 PH domain.

The mechanism of binding of two promising anticancer agents (the cytotoxic alkylphospholipids perifosine and miltefosine) to the Akt PH domain is investigated by high-resolution field-cycling (31)P nuclear magnetic resonance (NMR) spectroscopy using a spin-labeled recombinant PH domain. These results strongly indicate that there are two discrete amphiphile binding sites on the domain: (i) the cationic site that binds phosphoinositides and some alkylphospholipids and (ii) a second site that is occupied by only the alkylphospholipids. The identification of this second site for amphiphiles on the Akt1 PH domain provides a new target for drug development as well as insights into the regulation of the activity of the intact Akt1 protein. The field-cycling NMR methodology could be used to define discrete phospholipid or amphiphile binding sites on a wide variety of peripheral membrane proteins.