Parekh, Davey B.; (2001) An investigation into the regulation of nPKCs by phosphorylation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Three phosphorylation sites in the catalytic domains of cPKC isoforms have been shown to have profound effects on activity. These are the 'T loop', 'TP' and 'hydrophobic' sites. Little is known of the regulation of phosphorylation at these sites. The investigations here elucidate some of the controls that act upon the equivalent sites in nPKCs. To establish a system for these studies, polyclonal antisera selective for the phosphorylated forms of these sites in two nPKC isoforms, nPKCδ and nPKCε, were characterised. These tools were then used to investigate which cell culture condition is most suitable for monitoring the signalling inputs to these proteins. All three phosphorylation events were blocked by the C1-domain inhibitor calphostin-C. Initial studies established that T loop phosphorylation was under the influence of PI3-kinase, based upon sensitivity to the inhibitor LY294002. It was also found that the TP sites in nPKCδ and nPKCε were sensitive to the PKC-catalytic site inhibitor bis-indolylmaleimide I, consistent with an autophosphorylation process. The hydrophobic site did not appear to be autophosphorylated, but was influenced by phosphorylation at the T loop site. Phosphorylation at the hydrophobic site was shown to be under the control of mTOR, and also sensitive to amino acid deprivation, consistent with mTOR control. Intact cell studies demonstrated the potential role of PKCζ as a PKC-hydrophobic site kinase. Signalling initiated by both growth factors and β1-integrin activation played a role in the phosphorylation of these PKC sites. Cells had to be chronically deprived of both stimuli to reach a low basal phosphorylation at the T loop and hydrophobic sites in these nPKC isoforms. Interestingly, the tumour suppresser, PTEN, controlled the suspension-dependent behaviour of these phosphorylation sites. The complex behaviour of these PKC phosphorylations, their mutual interactions, effects upon activity and the implications for cell behaviour are discussed.

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