Jethwa, N; (2014) The role of polyphosphoinositides in Akt/PKB activation dynamics in subcellular compartments. Doctoral thesis , UCL (University College London).

Abstract

The phosphonoinosites play structural and functional roles in biological membranes; reversible phosphorylation of the inositol headgroup generates seven different headgroups which are involved in defining organelle identity and regulating signalling, trafficking and cytoskeletal integrity. The serine/ threonine kinase Akt lies at the heart of cell biology. It is activated by an interaction with the phosphoinositide PtdIns(3,4,5)P3 in response to growth factor stimulation. Akt controls cell growth, proliferation, survival and energy metabolism. Misregulated Akt activity is a hallmark of many different types of cancer and is a prominent feature of diabetes and obesity. A more detailed understanding of the mechanisms of Akt activation will be vital for the development of effective therapeutic interventions. Despite reports of the existence of phosphoinositides in intracellular compartments, it was thought that the only relevant site of Akt activation is PtdIns(3,4,5)P3 at the plasma membrane. In this thesis we took an interdisciplinary approach to assess the importance of intracellular membranes on Akt activation. We first optimised a drug inducible dimerisation system to acutely deplete PtdIns(3,4,5)P3 at the plasma membrane, leaving subcellular compartments untouched. We used HPLC coupled to tandem mass spectrometry to quantify cellular phospholipid composition, and developed a probe to detect PtdIns(3,4,5)P3 localisation by electron microscopy. We then used a FRET-based probe to monitor Akt localisation and conformation in plasma membrane-PtdIns(3,4,5)P3 depleted cells. Finally we used the inducible dimerisation system to recruit Akt to the early endosomes and nuclear envelope in order to determine whether localised Akt activation can take place away from the plasma membrane. We have shown for the first time that Akt interacts with PtdIns(3,4,5)P3 in subcellular compartments, resulting in localised activation and substrate phosphorylation; this suggests that intracellular Akt activation contributes to substrate specificity and signal duration, and may potentially be disrupted or upregulated in human disease.

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