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Cloning and characterisation of the PRK family: A novel family of protein kinases related to the PKC superfamily

Palmer, Ruth Helen; (1996) Cloning and characterisation of the PRK family: A novel family of protein kinases related to the PKC superfamily. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The work in this thesis describes the identification, cloning and characterisation of the PRK (PKC-related kinase) family of kinases. Initially this project was intended to clone a novel PKC isoform which had previously been biochemically identified in cell extracts. This objective led to the design of a degenerate PCR approach which exploited highly conserved regions within the PKC kinase domain which were not preserved throughout other serine/threonine kinases. Two PCR based screens were then carried out, the first from a U937 cDNA library and the second from a human fetal brain cDNA library. Analysis of the PCR products produced identified several novel sequences. Among these were three sequences - PRK1, 2 and 3 - which appeared to define a novel family of kinases, which while highly related to PKC seemed to be distinct. The full length cDNA sequences for PRK1 and PRK2 were then cloned and sequenced. Both encoded proteins in the 120-130 kDa range, with kinase domains closely related to the kinase domain of PKC and large amino-terminal 'putative' regulatory domains. On transient transfection both PRK1 and PRK2 expressed protein products of the expected size, which were capable of kinase activity (as measured by autophosphorylation) in anti-PRK1/2 immune precipitates. PRK1 protein was then overexpressed in COS 7 cells and purified to apparent homogeneity in order to carry out basic characterisation. The purified PRK1 protein migrated as a single polypeptide of mass 120 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. PRK1 has a substrate specificity that in part resembles that of protein kinase C (PKC), however unlike PKC, PRK1 was not activated by any combinations of phorbol esters, diacylglycerol and Ca2+. Nevertheless PRK1 was activated by limited proteolysis indicating a negative regulatory role for the amino-terminal domain(s) and suggesting that PRK1 may be regulated in vivo by an allosteric effector. Subsequent analysis revealed that PRK1 could be activated in vitro by various phosphoinositides, and more potently by the polyphosphoinositides PtdIns(4,5)P2 and Ptdlns(3,4,5)P3. Whether such activation actually occurs in vivo remains to be determined. The phosphorylation of substrates by PRK1 was also investigated, revealing that both the lipid kinase PtdIns-3-kinase and the cytoskeletal protein MARCKS (myristoylated alanine rich C kinase substrate) were phosphorylated in vitro by PRK1. Two-dimensional mapping of PRK1 phosphorylated MARCKS identified these sites as identical to the previously reported sites in MARCKS phosphorylated by PKC-. To try and assess the function of PRK1 in the intact cell stable selected cell lines were developed by transfection of a myc tagged PRK1 into NIH3T3 cells. By electron microscopy overexpression of PRK1myc in NIH3T3 fibroblasts results in the formation of vesicles of as yet undefined nature close to the plasma membrane. These results and the potential implications are discussed throughout this thesis.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Cloning and characterisation of the PRK family: A novel family of protein kinases related to the PKC superfamily
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10104082
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