The localisation and regulation of phosphatidylinositol-4-phosphate 5-Kinase gamma splice variants and the discovery of a new mammalian splice variant, PIP5KIγ_v6
University of Cambridge
Department of Pharmacology
Shandong Yingcai University
Doctor of Philosophy (PhD)
MetadataShow full item record
Xia, Y. (2011). The localisation and regulation of phosphatidylinositol-4-phosphate 5-Kinase gamma splice variants and the discovery of a new mammalian splice variant, PIP5KIγ_v6 (Doctoral thesis). https://doi.org/10.17863/CAM.15898
Type I PIP kinases (phosphatidylinositol 4-phosphate 5-kinases, PIP5Ks) catalyse the majority of cellular synthesis of PI(4,5)P2. To date, three mammalian isoforms (r1, r2, r3) have been found. PIP5KIr is subject to complex C-terminal splice variation, enhancing its transcriptional diversity through evolution and producing at least 5 known spliceoforms in the mammals. This study addresses several important questions. (1) Several remarkable differences have been discovered between the neuronal splice variant PIP5KIr_i3 and its close variant, Ir_i2, whose peptide lacks a 26-AA insert near its C-terminus. This study attempts to map these behavioural differences onto motifs within the peptide insert. Furthermore, a site of point mutation is identified near the activation loop, which amplifies the above differences. (2) This study documents properties of the more recently discovered PIP5KIr_i3, about which relatively little is known, for example, the regulation of its subcellular localisation, kinase activity and post-translational modifications. By site-directed mutagenesis and examining more closely several crucial motifs, insight is gained into the putative relationship between the enzyme’s phosphorylation state, cellular localisation, lipid kinase activity and autophosphorylation. (3) The discovery of a new PIP5KIr splice variant, Ir_v6, is described. First discovered in rodents, PIP5KIr_i6 encompasses the 26-AA insert of Ir_i3, but lacks the common C-terminus of Ir_i2 and Ir_i3 which contains peptide motifs that have several roles in vivo. A polyclonal antibody against the C-terminus of Ir_i6 was also developed. Preliminary characterisation of Ir_i6 demonstrates a similar subcellular localisation, but a wider expression profile than its close relative, Ir_i3, suggesting potentially differential functions across tissues and at various developmental stages. (4) The existence of Ir_v3 and Ir_v6 is also confirmed in humans. In light of recent findings of other novel human spliceoforms, this is shown to be a case of intra-exonic splicing producing “alternative 5’ splice site” exons in the human. Overall, this thesis should help to better understand the regulation and physiological roles of PIP5KIr and, specifically, its different splice variants.
Phosphatidylinositol 4-phosphate 5-kinase I gamma, Type I PIP5K, Phosphorylation, Alternative splicing, Discovery, Subcellular localisation, Confocal microscopy, Polymerase chain reaction, Kinase assay, Autophosphorylation
This work was supported by the Dorothy Hodgkin Postgraduate Award, Trinity College Cambridge and Shandong Yingcai Education Trust, China.
This record's DOI: https://doi.org/10.17863/CAM.15898
All the experiments described in this dissertation were carried out solely by the author unless otherwise stated, in the Department of Pharmacology, University of Cambridge, between January 2008 and September 2010. This dissertation is not substantially identical to any other submitted for a degree, diploma or other qualifications, and no part of it has been, or is currently being, submitted for any such degree, diploma or other qualifications. This dissertation does not exceed the prescribed limit of 60000 words.