Proteomic investigation of the class IA phosphoinositide 3-kinase signalling pathway

PhDClass IA phosphoinositide 3-kinases (PI3Ks) are a family of enzymes with key roles in the regulation of signalling pathways, many of which are mediated through Akt. The PI3K/Akt pathway has a critical role in the regulation of key cellular functions such as cell survival, growth, proliferation, migration and metabolism and is involved in several diseases. Advances in mass spectrometry and separation sciences have revolutionized the field of proteomics. Techniques based on mass spectrometry are now the first choice for protein identification and are increasingly important for their quantitative analysis. The development of phosphoproteomics has also permitted the global quantitative analysis of signalling activity. The aim of this project was to contribute to our understanding of the activity and regulation of the PI3K/Akt signalling pathway from a proteomics perspective. Novel approaches based on mass spectrometry were designed, developed and applied to the investigation of this signalling pathway. Firstly, we investigated the hypothesis that PI3K activity may be regulated by dynamic protein interactions. We designed an affinity purification mass spectrometry strategy to identify proteins interacting dynamically with PI3K. Our study revealed that calpain small subunit 1 interacts dynamically with PI3K. Further investigation demonstrated that active calpain heterodimers associate dynamically with PI3K, thereby regulating PI3K stability and activity. Secondly, we characterized phosphorylation events downstream of Akt, a major downstream effector of PI3K. We used global shotgun phosphoproteomics of a cell line expressing an inducible construct encoding constitutively active Akt to identify phosphorylation events downstream of Akt in vivo. In addition, we also developed an in vitro kinase assay which, when coupled to global shotgun phosphoproteomics, enables the quantification of Akt1 activity in addition to the identification of downstream phosphorylation sites. Furthermore, we found that this in vitro approach may be used as a method suitable for the global profiling of endogenous kinase activities.Medical Research Counci