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The involvement of PKD in Fcγ receptor activation of the NADPH oxidase in human neutrophils

Davidson-Moncado, Jan Kenneth; (2000) The involvement of PKD in Fcγ receptor activation of the NADPH oxidase in human neutrophils. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

NADPH oxidase is a membrane bound enzyme, crucial in the destruction of pathogens by neutrophils. We have studied the signalling cascade leading to the activation of the NADPH oxidase upon activation of the Fc receptor, with a particular focus on the involvement of protein kinases in this process. Activation of the NADPH oxidase following Fcγ receptor stimulation was studied in human neutrophils using inhibitors of tyrosine kinases, phosphatidyl inositol 3-kinase and protein kinase C (PKC), which are all involved in this signalling cascade. Results with different PKC inhibitors showed unexpected results. While Go 6976 inhibited activation completely, Ro 31-8220 caused only a 60% inhibition. Thus a Go 6976-sensitive Ro 31-8220-insensitive component was suspected. Evidence from the literature suggest that Go 6976 but not Ro 31-8220 inhibits an isotype of the PKC family, PKCµ/Protein kinase D (PKD), in vitro, indicating that PKD could be involved in NADPH oxidase activation. PKD antisense oligonucleotides inhibited NADPH oxidase activation, supporting an involvement of PKD. Kinase assays showed that PKD is activated upon Fc receptor ligation. Immunofluorescence techniques showed that PKD is present in the plasma membrane and appears in the phagosomal membrane upon phagocytosis, further linking its functions to NADPH oxidase activation. The NADPH oxidase contains the membrane bound proteins, gp22phox and gp91phox and the cytosolic components, p47phox, p40phox and p67phox, which translocate to the phagosomal membrane upon activation. Translocation of these cytosolic components is driven by phosphorylation. We have shown that PKD phosphorylates p40phox and p47phox but not p67phox. Mass spectrometry studies and 2 dimensional gel electrophoresis technology indicated that PKD may not only play a role in the activation of the NADPH oxidase, as described, but may also phosphorylate other proteins, including some involved in cytoskeletal function, signalling and metabolism. The further characterisation and the roles of these need investigation. Thus PKD is present in neutrophils and is activated upon Fc receptor stimulation. It also plays a role in the signalling cascade that leads to NADPH oxidase.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The involvement of PKD in Fcγ receptor activation of the NADPH oxidase in human neutrophils
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Protein kinase D
URI: https://discovery.ucl.ac.uk/id/eprint/10107112
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