Photo-Activatable Akt Probe - A New Tool to Study the Akt-Dependent Physiopathology of Cancer Cells.
Abstract:
Akt is commonly overexpressed and activated in cancer cells, and plays a pivotal role in cell survival, protection andchemo-resistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anti-cancer drugs. Here, we examined whether a newly developed photo-activatable Akt (PA-Akt) probe, based on a light-inducible protein interaction module of plant cryptochrome2 (CRY2) and cryptochrome-interacting basic-helix-loop-helix (CIB1), can regulate Akt-associated cell functions. By illuminating blue light to the cells stably transfected with PA-Akt probe, CRY2-Akt (a fusion protein of CRY2 and Akt) underwent structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1) anchoring at cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probe-Akt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3 , one of the main downstream signals of Akt, were also phosphorylated, depending on light intensity. These facts indicate that photo-activation of probe-Akt can activate endogenous Akt and its downstream signals. The photoactivated Akt conferred protection against nutritional deprivation and H2O2 stresses to the cells significantly. Using the newly developed PA-Akt probe, endogenous Akt was activated easily, transiently and repeatedly. This probe will be a unique tool in studying Akt-associated specific cellular functions in cancer cells and developing anti-cancer drugs.
