Open Access

ARTICLE

Photo-Activatable Akt Probe: A New Tool to Study the Akt-Dependent Physiopathology of Cancer Cells

Sanae Haga^*, Takeaki Ozawa^†, Naoki Morita^‡, Mami Asano^§, Shigeki Jin^¶, Yimin^#, Michitaka Ozaki^*§¶

* Department of Biological Response and Regulation, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan
† Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan
‡ Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan
§ Laboratory of Molecular and Functional Bioimaging, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan
¶ Core Research Laboratory, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan
# Department of Advanced Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Japan

Oncology Research 2018, 26(3), 467-472. https://doi.org/10.3727/096504017X15040166233313

Abstract

Akt is commonly overexpressed and activated in cancer cells and plays a pivotal role in cell survival, protection, and chemoresistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anticancer 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 a structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1), anchoring it at the cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probeAkt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3b, 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 photo-activated 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 anticancer drugs.

---

Keywords

---