Panaretou, Christina; (1998) Studies of a phosphatidylinositol 3-kinase complex linked to vesicular trafficking in human cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphoinositide 3-kinases (PI 3-kinases) are signal transducing molecules that catalyse the addition of phosphate to the 3-OH position of the inositol ring of phosphoinositides. PI 3-kinase has emerged as an important mediator in the events leading to mitogenesis, cytoskeletal re-arrangement, receptor internalisation and glucose transport. In mammalian cells numerous PI 3-kinases have been discovered, including those regulated by Receptor Tyrosine Kinases (RTK) and heterotrimeric G proteins. These PI 3-kinases can generate Phosphatidylinositol (PtdIns)3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 as potential second messengers. In contrast, genetic and biochemical studies have shown that yeast contain only one PI 3-kinase, termed Vps34p. This enzyme uses PtdIns as its only substrate and is not activated by any known ligand. Vps34p is found associated with Vps15p, a 170kDa serine/threonine protein kinase. This heterodimeric complex is required for the efficient sorting and delivery of proteins to the yeast vacuole. A human homologue of the yeast Vps34p protein, PtdIns 3-kinase has been identified and shown to form a complex with a protein of 150kDa (termed p150), raising the possibility that a human Vpsl5p homologue exists. In this study the cDNA encoding the p150 protein was isolated and used to express a 150kDa protein in insect (Sf9) and mammalian (COS-7) cells, p150 was shown to display extensive amino acid homology with Vps15p and sequence comparisons between both proteins have been used to delineate potential functional domains. The data presented in this thesis also show that recombinant p150 associates with PtdIns 3-kinase both in vitro and in vivo. Using immunofluorescence techniques, p150 expressed in COS-7 cells was localised to perinuclear punctate compartments. To investigate which of the p150 domains are responsible for its subcellular localisation and association with PtdIns 3-kinase, deletion and amino acid substitution studies were undertaken. Additional biochemical studies investigating the 3'-phosphoinositides produced in cells expressing PtdIns 3-kinase demonstrated elevated levels of PtdIns3P and PtdIns(3,5)P2. The increase in PtdIns3P production by the p150/PtdIns 3-kinase complex in the presence of phosphatidylinositol transfer protein (PI-TP) also suggests a role for PI-TP in PtdIns substrate presentation to PtdIns 3-kinase. The results presented in this study demonstrate that p150 is the human Vps15p homologue, suggesting an evolutionary conservation between yeast and humans in the basic molecular mechanisms of vesicle transport. Based on the data presented, mechanisms by which the p150/PtdIns 3-kinase complex and its 3'-phosphoinositide products might be implicated in membrane trafficking events are proposed.

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