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Title: Determination of the functional regions involved in WNK1 and WNK4 regulation of NCCT and ROMK membrane expression
Author: Cope, G.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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Abstract:
Mutations in the with no lysine family of serine/threonine kinases WNK1 and WNK4 are known to cause Gordon’s syndrome. At least two further genetic loci have not been assigned candidate genes. One of these loci has been mapped to q32-42 region on chromosome 1; this subset is known as PHA2A. Mutation scanning of two candidate targets PTPN14 and Synaptotagmin 2, within kindred suspected of harbouring the chromosome 1 defect, failed to identify any disease-causing mutation. All did possess however an Ile 3999 Arg substitution within the Synaptotagmin 2 gene. Analysis of 50 control samples showed that 47% of control subjects carried at least one allele, suggesting this is a very common SNP unconnected with Gordon’s syndrome. Initial work has indicated a role for WNK4 in inhibiting sodium/chloride reabsorbtion through NCCT; this effect is absent in WNK4 containing a typical disease causing mutation or by the coexpression of WNK1. It has been proposed that WNK4 kinase activity is necessary for the action of WNK4 together with a C-terminal association with NCCT and that WNK1 acts through protein-protein mediated interaction with WNK4 removing its inhibitory action on NCCT. Using either truncated WNK4 proteins 1-620 amino acid (containing the kinase domain and acidic motif) or 620-end (coiled-coil and proteine rich region), I demonstrate that the WNK4 interaction with NCCT is mediated through the initial 620 amino acids including the kinase domain, and that the remaining portion has no direct effects on NCCT when expressed in Xenopus oocytes. This interaction is probably indirect as immunoprecipitation using the affinity tag expressed on the truncations failed to associate with NCCT as assessed by blotting with anti-NCCT. Expression of further site-mutated constructs mimicking the Gordon’s proteins and a variant in which the acidic motif was deleted (ΔAM) were used to determine the role of this motif in the interaction. Both mutant WNK4 proteins removed inhibition of WNK4, while a charge neutral mutant maintained inhibition. This suggests that the interaction is mediated through protein-protein interactions at this region and that electrostatic charge is a key part of this interaction. Expression of catalytically inactive WNK1 is also able to remove inhibition of WNK4 suggesting the WNK1 effect is independent of its kinase activity. Expression of truncated variants of WNK1, 1-501 containing the kinase domain 502-1100 acidic motif and coiled coil and 1101-end CT truncation all are unable to remove inhibition of WNK4 suggesting a fully intact WNK1 protein is necessary to effect removal of inhibition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.597982  DOI: Not available
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