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Regulation of the protein kinase C-related kinase family

Flynn, Peter; (1999) Regulation of the protein kinase C-related kinase family. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Protein phosphorylation and its control by the action of protein kinases and phosphatases has been shown to play an essential role in signal transduction pathways and hence cell functions. More recent discoveries have implicated GTP hydrolysing enzymes and lipids; adding to the depth of complexity required for the balance and amplification of signals. The Protein Kinase C (PKC) family of kinases can be subdivided based on their requirement for Ca2+, Diacylglycerol (DAG) and phosphor-lipids. Previously, a PCR-based screen for novel PKC isoforms produced two new gene products, which were subsequently fully cloned: Protein Kinase C-Related Kinases (PRK 1 and 2). These were seen to be highly homologous to the PKCs in their kinase domains but had novel N-terminal regulatory regions. Their serine/threonine kinase activity was insensitive to both Ca2+ and DAG but was activated in vitro by limited proteolysis and polyphosphoinositides Ptdlns(4,5)P2 and Ptdlns(3,4,5)P3. The aim of this thesis was to investigate the control of the PRKs. Initial work focused on the potential interaction between the PRKs and Small GTPases of the Rho family, and assessed what role such an interaction would have on the kinase's activity. A variety of in vitro techniques were used to characterise the site of contact on both the PRKs and the Rho proteins. The nucleotide-specificity of binding was also analysed. The definition of the Rho binding region within the PRKs novel regulatory region (Homology Region 1) suggested the GTPase disrupted an intramolecular, pseudo-substrate site interaction with the kinase domain. However, our data showed only a two-fold increase in the PRK's kinase activity when co-expressed in cultured cells with an active Rho. Nevertheless we were able to show translocation of PRK to early endosomes with RhoB overexpression and further that a hyperphosphorylated form of the kinase existed within this sub-cellular compartment. This suggested that other components were involved in the control of the kinase. On consideration of the similarities between members of the AGO kinase subfamilies within the activation-loop, the involvement of the upstream kinase PDK1 in PRK activation was assessed. Co-expression studies in cultured cells allowed the definition of a Rho-dependent interaction between the two kinases and a three-way co-localisation of PRK, RhoB and PDK1 on early endosomes. An activation loop phosphorylation event was analysed using phospho-specific polyclonal antisera. In vitro and in vivo studies using both wild type and mutant PRKs demonstrated that this phosphorylation event was necessary for kinase activity. It has thus been shown that both Rho family GTPases and PDK kinases, which depend on the products of PI3-Kinase activity, are required for the cellular localisation and activation of the PRK sub-family of kinases.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of the protein kinase C-related kinase family
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Protein phosphorylation
URI: https://discovery.ucl.ac.uk/id/eprint/10120556
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