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dc.contributor.advisorMcColl, Shaun Reuss-
dc.contributor.advisorKlingler-Hoffmann, Manuela-
dc.contributor.authorTurvey, Michelle Elizabeth-
dc.description.abstractThe Class IB phosphatidylinositol 3-kinase (PI3K) enzyme, PI3Kγ, is activated and recruited to the plasma membrane in response to G protein-coupled receptor stimulation. Upon activation, the lipid-kinase activity and downstream signalling cascades initiated by PI3Kγ lead to cytoskeletal rearrangements and the formation of a leading edge for the induction of directed cell migration. PI3Kγ consists of the catalytic subunit p110γ, which forms a mutually exclusive heterodimer with one of two regulatory adaptor subunits, p84 or p101. Although expressed by most cells in the organism, PI3Kγ subunits are expressed at highest levels in motile haematopoietic cells, where the regulation of PI3Kγ signalling is critical to controlling and maintaining coordinated cell migration during immune responses. Consistent with a central role in leukocyte chemotaxis, innate and adaptive immune cell subsets from p110γ-deficient mice have been shown to exhibit migration defects in vitro and in vivo. Furthermore, the aberrant expression of PI3Kγ subunits and dysregulation of PI3Kγ signalling pathways has been shown to contribute to pathologies such as cancer and autoimmunity where enhanced cell migration promotes disease progression. Despite this, the mechanistic basis for PI3Kγ signal regulation is not well understood, particularly with respect to the distinct contributions of the individual regulatory adaptor subunits, p84 and p101. Many PI3Kγ-dependent cell functions have been elucidated experimentally using p110γ- and p101-deficient genetically-modified mouse strains and the PI3Kγ-selective inhibitor, AS605240. However, detailed functional data regarding p84 is lacking due to the absence of a p84-deficient mouse strain and limited availability of high quality p84-specific reagents. Three major research goals were addressed in the present study to improve our understanding of the role of p84 in PI3Kγ lipid-kinase signalling and its implication in PI3Kγ-dependent cell migration. The first goal was to examine the phosphorylation status of p84 during PI3Kγ signalling and assess the role of identified regulatory phosphorylation sites for p84 function using the mammary epithelial carcinoma model cell line, MDA.MB.231. Data presented in this thesis demonstrate that in contrast to the p110γ and p101 subunits that promote the migration and metastasis of carcinoma cells, the p84 adaptor protein has tumour suppressor function in vitro and in vivo, which was determined to be dependent on a potential phosphorylation site within p84, Thr607. It was found that Thr607 was required for p84 to form an inducible heterodimer with p110γ (after initial PI3Kγ signal activation) in a complex sequestered from active signalling at the membrane. This Thr607-dependent p84/p110γ dimerisation may therefore represent a novel mechanism of negative PI3Kγ signal regulation that limits the migration and metastasis of cancer cells. Next, the contribution of p84 to PI3Kγ-dependent immune cell function was determined through the generation and characterisation of a novel p84-deficient mouse (Pik3r6⁻ʹ⁻) using CRISPR gene-editing technology. Pik3r6⁻ʹ⁻ mice were characterised in the context of immune cell development, activation and migration in a variety of haematopoietic cell subsets. It was shown that Pik3r6⁻ʹ⁻ mice develop normally with respect to lymphoid organ and circulating leukocyte populations at homeostasis. However upon stimulation, neutrophils from Pik3r6⁻ʹ⁻ mice display reduced migration in response to GPCR agonists in vitro and in a murine model of inflammatory autoimmunity (experimental autoimmune encephalomyelitis; EAE), it was found that activated Th lymphocytes display impaired trafficking and reduced infiltration to inflammatory sites. The final goal was to develop and optimise a proteomic platform to investigate and compare the proteomes of migratory CD4⁺ lymphocytes isolated from tissues at different stages of inflammatory disease progression using experimental autoimmune encephalomyelitis as a model. An isotope-coded protein-labelling (ICPL) approach was developed and optimised to assess the proteomes of CNS-infiltrating CD4⁺ lymphocytes during disease progression in two models of EAE; chronic MOG₃₅₋₅₅-induced EAE and relapsing-remitting PLP₁₃₉₋₁₅₁-induced EAE. This study identified differentially regulated proteins related to immune cell function and represented a initial feasibility study to verify the validity of ICPL as an approach to examine the differential proteomes of wildtype and p84-deficient migratory CD4⁺ lymphocytes during inflammatory disease. Collectively, the data presented in this thesis represent the identification and characterisation of novel roles for p84 within PI3Kγ lipid-kinase signalling during both the regulation of cell migration in carcinoma cells and in haematopoietic cells during immune responses. In addition to furthering the understanding of the unique roles for p84 within PI3Kγ signal regulation, the generation of a p84-deficient mouse strain constitutes an important tool to further experimental research in this area.en
dc.subjectp84 adaptor subunit of PI3Kγen
dc.subjectP13K signallingen
dc.subjectcell migrationen
dc.titleThe role and regulation of the p84 adaptor subunit in phosphatidylinositol 3-kinase γ lipid-kinase signalling and the control of PI3Kγ-dependent cell migrationen
dc.contributor.schoolSchool of Biological Sciencesen
dc.provenanceCopyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.en
dc.provenanceThis electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
dc.description.dissertationThesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015.en
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