Renal COP9 Signalosome Deficiency Alters CUL3-KLHL3-WNK Signaling Pathway

• Published in: Journal of the American Society of Nephrology 2018-11, Vol.29 (11), p.2627-2640
• Main Authors: Cornelius, Ryan J, Si, Jinge, Cuevas, Catherina A, Nelson, Jonathan W, Gratreak, Brittany D K, Pardi, Ruggero, Yang, Chao-Ling, Ellison, David H
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Animals; Basic Research; COP9 Signalosome Complex - deficiency; COP9 Signalosome Complex - genetics; COP9 Signalosome Complex - metabolism; Cullin Proteins - metabolism; Disease Models, Animal; Female; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Kidney - metabolism; Kidney - pathology; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins - metabolism; Microscopy, Fluorescence; Mutation; Nephrons - metabolism; Nephrons - pathology; Peptide Hydrolases - deficiency; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Phenotype; Protein Serine-Threonine Kinases - metabolism; Proteolysis; Pseudohypoaldosteronism - genetics; Pseudohypoaldosteronism - metabolism; Pseudohypoaldosteronism - pathology; Signal Transduction
• ISSN: 1046-6673; 1533-3450
• DOI: 10.1681/ASN.2018030333

The familial hyperkalemic hypertension (FHHt) cullin 3 (CUL3) mutant does not degrade WNK kinases normally, thereby leading to thiazide-sensitive Na-Cl cotransporter (NCC) activation. CUL3 mutant (CUL3 9) does not bind normally to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin-RING ligases. CUL3 9 also caused increased degradation of the CUL3-WNK substrate adaptor kelch-like 3 (KLHL3). Here, we sought to determine how defective CSN action contributes to the CUL3 9 phenotype. The Pax8/LC1 mouse system was used to generate mice in which the catalytically active CSN subunit, , was deleted only along the nephron, after full development (KS- ). Western blot analysis demonstrated that deletion increased the abundance of neddylated CUL3. Moreover, total CUL3 expression was reduced, suggesting decreased CUL3 stability. KLHL3 was almost completely absent in KS- mice. Conversely, the protein abundances of WNK1, WNK4, and SPAK kinases were substantially higher. Activation of WNK4, SPAK, and OSR1 was indicated by higher phosphorylated protein levels and translocation of the proteins into puncta, as observed by immunofluorescence. The ratio of phosphorylated NCC to total NCC was also higher. Surprisingly, NCC protein abundance was low, likely contributing to hypokalemia and Na and K wasting. Additionally, long-term deletion resulted in kidney damage. Together, the results indicate that deficient CSN binding contributes importantly to the FHHt phenotype. Although defective CUL3 9-faciliated WNK4 degradation likely contributes, dominant effects on KLHL3 may be a second factor that is necessary for the phenotype.