Das, Pamela; (1998) Complex interactions of the domains of the p85 adaptor subunit of phosphoinositide 3-kinase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes studies of signal transduction in mammalian systems which are designed to help understand how molecules interacting at the cell surface regulate intracellular responses. In recent years, it has become clear that phosphoinositides (PI) are key components of growth factor triggered signal transduction cascades, and this thesis describes work to elucidate the structure and function of members of the Phosphoinositide 3-kinase (PI3K) family of enzymes. The p85 adaptor subunit of PI3K contains several domains including SH3 (Src homology domain 3), SH2 (Src homology domain 2) and BH (Breakpoint cluster region homology). This thesis describes work which examines the relationship between the structure and function of these domains. As part of the study, a number of deletions and point mutations were introduced into the p85 cDNA. The encoded proteins were expressed either in bacteria (E.coli), insect (Sf9) and mammalian (COST) cells, and the structure and function of the intact p85 and its isolated domains were examined using biochemical and biophysical techniques. To investigate the effects of the mutations on the regulation of the enzymatic complex, specific studies involving size-exclusion chromatography of the recombinant proteins, identification of associated proteins using affinity chromatography, biosensor analysis to establish binding affinities, and lipid and protein kinase assays of the recombinant enzymatic complex were used. This study presents data and describes proposals which clarify the possible contributions of the SH3 and BCR domains to the function of the p85 adaptor subunit of PI3K. The results presented here show how the SH3 and BCR domains associate with other regions of p85 in vitro, and mechanisms are proposed to explain interactions of p85 with other proteins involved in signal transduction in vivo. Finally based on the data presented, mechanisms by which this enzyme is activated and regulated downstream of transmembrane receptors, are proposed.

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