Srivastava, Jyoti; (2002) Regulation of protein kinase C delta degration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Reversible phosphorylation of proteins is a vital means of regulation in signal transduction processes. The PKC family of lipid dependent serine/threonine kinases is known to play key roles in a whole range of cellular functions. To comprehend the diverse roles of the different family members, it is important to define the regulation of each PKC isoform. While activation of PKC isoforms is critical for their function in targeting downstream substrates/effectors, of equal importance is the desensitisation of the active kinases in determining their consequential effects. The overall aim of this thesis was to explore the mechanism(s) causing the downregulation and degradation of PKCδ. The degradation of this novel isoform of PKC has been examined in NIH 3T3 cells, in response to chronic activation by the phorbol ester TPA. A significant part of this study focused on the dephosphorylation of PKCδ as a primary event predisposing the enzyme to degradation. This was addressed in vivo using cell permeable inhibitors of protein phosphatases. In addition, in vitro studies using purified forms of PP1, PP2A and PP2C were carried out to investigate the potential effects of the different protein phosphatases towards PKCδ as a substrate. Consistent with other data, PP2A was identified as the putative protein phosphatase involved in the dephosphorylation of PKCδ. Interestingly, studies revealed that either nucleotide binding or substrate binding to PKCδ protected the enzyme from the action of phosphatases. To further investigate the requirement of phosphorylation/dephosphorylation during the downregulation process in cells, phosphorylation site mutants of PKCδ were generated and studied in transient transfection experiments. While these provided useful information, it was concluded that ectopic expression of PKCδ did not entirely represent the behaviour of the endogenous protein. Nevertheless, information from mutant analyses together with data from in vivo studies of endogenous PKCδ, in contrast to the previous speculation, provides strong evidence for the role of phosphorylation in the targeting of PKCδ to degradation via the ubiquitin/proteasome pathway.

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