Structural Basis for Akt Translocation to the PM in Breast Cancer Cells

Abstract

Akt is a serine/threonine kinase that plays a key role in the regulation of cell survival, apoptosis and oncogenesis. The translocation of Akt to the plasma membrane (PM) for phosphorylation and subsequent activation is a critical step in the Akt activation pathway. It has been established that membrane binding of Akt is mediated by direct interactions between it's pleckstrin homology domain (PHD) and lipids such as phosphatidylinositol‐(3,4,5)‐trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in breast cancer cells is also modulated by the calcium‐binding protein, calmodulin (CaM). Upon epidermal growth factor (EGF) stimulation of these cells, CaM co‐localizes with Akt at the PM to promote membrane anchoring and activation. Although previous studies have shown that both PI(3,4,5)P3 and CaM may modulate Akt activation, the molecular mechanisms governing these interactions have not been well characterized. We hypothesize that PI(3,4,5)P3 and CaM act in synergy by binding to Akt(PHD) simultaneously to facilitate anchoring to the PM. In this study we employ biochemical, biophysical and nuclear magnetic resonance (NMR) techniques to characterize the interaction between CaM and PI(3,4,5)P3 to Akt(PHD). Our results demonstrate that there is a direct interaction between CaM and Akt(PHD) and suggest that Akt(PHD) undergoes significant conformational changes upon CaM binding. We also provide evidence that both PI(3,4,5)P3 and CaM bind to Akt(PHD) simultaneously. Elucidation of the structural basis for Akt‐CaM and Akt‐PI(3,4,5)P3 interaction is critical for understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.