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The structure and function of human phosphoinositide 4-kinases

Minogue, Shane; (1999) The structure and function of human phosphoinositide 4-kinases. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The biosynthesis of phosphosphoinositides underlies a wide range of important biological processes including receptor signalling, cytoskeletal remodelling, and vesicle traffic. This thesis describes the molecular cloning and biochemical characterisation of phosphatidylinositol 4-kinaseβ (Ptdlns 4Kβ) and the type IIα phosphatidylinositol(5)phosphate 4-kinase (PtdInsPK IIα), two enzymes believed to lie in PtdIns(4,5)P2 biosynthetic pathways. I have cloned and characterised two splice variants of a novel wortmannin-sensitive, cytosolic PtdIns kinase, PtdIns 4Kβ, with homology to Pik 1p, the archetypal Ptdlns 4K from S. cerevisiae. The two PtdIns 4Kβ proteins differ by the presence of a short serine- rich insertion which can be phosphorylated in vitro by casein kinase II. Also presented in this thesis is the cloning of PtdInsPK IIα and its expression in E. coli, S. frugiperda and mammalian cells. Recombinant protein has been used to produce a monoclonal antibody which recognises PtdInsPK IIα by western blotting, immunoprecipitation and immunofluorescence. Sequence analysis of PtdInsPK IIα showed no statistically significant homology with any known phosphotransferase enzyme. However, identification of the nucleotide-binding residue using a combination of affinity labelling and mass spectrometry has permitted identification of the active site. The importance of the nucleotide-binding residue, and of others predicted to be important in catalysis, has been confirmed by analysis of mutant proteins generated by site-directed mutagenesis. The unique substrate specificity of PtdInsPK IIα suggests that it may lie in a previously undescribed PtdIns(4,5)P2 biosynthetic pathway. The possible significance of this pathway is discussed.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The structure and function of human phosphoinositide 4-kinases
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Phosphosphoinositides
URI: https://discovery.ucl.ac.uk/id/eprint/10102553
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