Final Report Summary - BETA (How is phosphoinositide 3-kinase beta regulated by G-Protein Coupled Receptors and by Rab-5?)
To start my project, I first cloned, expressed and purified many PI3Kbeta constructs. Each construct was composed of the full-length catalytic subunit (p110beta) linked to a regulatory subunit (p85) where one to 4 individual domains were removed. This truncation strategy was used to try to identify the most stable constructs to increase chances to get a crystal structure. In parallel and thanks to collaboration with Prof. Bernd Nürnberg, we could get purified Gbetagamma heterodimers expressed in insect cells. The PI3Kbeta constructs were then individually mixed with purified Gbetagamma heterodimers and tested over >1700 different conditions for crystallization. After thorough investigations of every crystallization conditions, no crystals could be seen and I decided to use Hydrogen/Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) as an alternative technique to gain structural and dynamic information on the PI3Kbeta-Gbetagamma interaction. Using this powerful and novel technique, which is based on the exchange rate of amide protons with solvent, I mapped regions on PI3Kbeta that were involved in interactions with either Gbetagamma or lipid membranes. Based on the HDX-MS data, I could design a PI3Kbeta mutant that was no longer stimulated by Gbetagamma, but retained basal or growth factor stimulation mimicked activity untouched. Having identified such a Gbetagamma insensitive PI3Kbeta mutant proved then very useful for understanding the importance of this interaction for PI3Kbeta cellular function. Thanks to a fruitful collaboration with the group of Prof. Johathan Backer at Einstein College in New York, we established that the Gbetagamma-PI3Kbeta interaction was required for cell transformation, and that blocking of this interaction with a p110beta-mimicking peptide could reduce proliferation of cancer cells. Those results were published in December 2012 in Science Signaling, and the illustration showing part of our results was selected for cover image.
This work has characterized at the molecular level the interaction between Gbetagamma, which are released by activated GPCRs, and its effector PI3Kbeta. These findings have served as a basis for understanding the role of this interaction in cells, establishing the GPCR-Gbetagamma-PI3Kbeta signaling axis as essential for cell transformation. Blocking of the Gbetagamma-PI3Kbeta interaction with a p110beta-peptide was able to reduce proliferation of PTEN-null tumor cells, establishing the Gbetagamma-PI3Kbeta interface as a target cancer therapy.
Details about the project can be found on Roger Williams’ web site: http://www.mrc-lmb.cam.ac.uk/rlw/text/rlw_homepage/