Regulation of renal ion transport by the CUL3-WNK-SPAK pathway

Oregon Health & Science University, Portland OR

Investigators

Linked publications & trials

Abstract

Project Summary In the kidney WNK kinases activate the downstream kinases SPAK and OSR1 that directly phosphorylate the NaCl cotransporter NCC. In Familial or CUL3, produced Hyperkalemic Hypertension (FHHt) mutations i n WNKs, Cullin 3 (CUL3), KLHL3 lead to NCC hyperactivation. The Cullin Ring Ligase (CRL) complex, composed of the caffold the substrate adaptor KLHL3, and the ligase RING, degrades WNKs. The effects of mutant CUL3, by skipping of exon 9 that deletes 57 amino acids (CUL3-∆9), are complex and not completely s understood. CUL3-∆9 promotes its own degradation in vitro and in a mouse model of CUL3 FHHt. Our in vitro data support an additional effect of CUL3-∆9 to promote KLHL3 degradation exceeding that seen for wild type (WT) CUL3. Our in vivo data suggest that the combination of lower total CUL3 and KLHL3 plays a central role in NCC hyperactivation in CUL3-∆9-mediated FHHt. Recently, a novel FHHt-causing mutation was identified (CUL3474-477). While CUL3474-477 also promotes its own degradation in vitro our new preliminary data suggest it does not promote KLHL3 degradation. We hypothesize that this minimizes NCC dysregulation and may explain the milder electrolyte abnormalities presenting with CUL3474-477. Our preliminary data also suggest that KLHL3, but not other CUL3 substrate adaptors, displays sensitivity to CUL3-mediated degradation. We have identified a structural feature that may explain this KLHL3-specific effect. Our previous work has also shown that CUL3- ∆9 displays defects in a CRL regulatory process that involves covalent attachment (“neddylation”) and removal (“deneddylation) of NEDD8, a small ubiquitin-like protein. CUL3-∆9 cannot be deneddylated due to loss of interaction with the enzyme JAB1. Our data suggest this not only contributes to the etiology of FHHt, but also causes renal injury. However, the whether neddylation (as opposed to deneddylation) directly contributes to FHHt severity, and its role in in renal function, is unknown. Our overall aim is to determine the mechanisms underlying CUL3-∆9-mediated FHHt, and gain insight into the roles of the CRL and its regulatory system in the kidney. In Aim 1 we will determine the basis of the unique sensitivity of KLHL3 to degradation by WT CUL3, and the basis for enhanced KLHL3 degradation by CUL3-∆9 but not CUL3474-477, by performing structural homology modeling, transfection experiments, and biophysical assays to asses flexibility and binding. We will use global CUL3474-477 knockin-mice to determine the effects of CUL3474-477 on NCC activity and electrolyte homeostasis. We will also test the hypothesis that exon 9 splice mutations differ in their degree of mis-splicing potential, which may affect the amount of CUL3-∆9 generated and hence KLHL3 degradation. We will thus determine the precise molecular mechanisms through which CUL3-∆9 causes FHHt, and explain the heterogeneity of FHHt severity seen with different CUL3 mutations. In Aim 2, we will determine the contribution of neddylation to FHHt by disrupting NAE1, a key enzyme required for addition of NEDD8 to CUL3, genetically and pharmacologically. We will also genetically disrupt NAE1 to determine its roles in renal function.