Structural and Biophysical Characterization of the Interactions Between Calmodulin and the Pleckstrin Homology Domain of Akt [Molecular Biophysics]
September 21st, 2015 by Agamasu, C., Ghanam, R. H., Saad, J. S.
The translocation of Akt, a serine/threonine kinase, to the plasma membrane (PM) is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon epidermal growth factor (EGF) stimulation of breast cancer cells, CaM co-localizes with Akt at the PM to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed nuclear magnetic resonance (NMR), biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PH domain of Akt (dissociation constant = 100 nM). The interaction between CaM and Akt(PHD) is enthalpically driven and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for the interaction. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P3 binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P3 upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.