The Nedd8 Co‐E3 DCNL4 is regulated by dietary potassium in renal intercalated cells and promotes WNK kinase degradation via the KLHL3/CUL3 complex

Abstract

WNK kinases regulate NaCl and K+ transport in the distal nephron. The abundance of these kinases is controlled by the KLHL3/CUL3 complex, a cullin‐RING E3 ligase (CRL) that ubiquitylates the WNKs, marking them for degradation. Little is known about the mechanisms that physiologically regulate KLHL3/CUL3 complex activity. Mammalian DCN‐like (DCNL) proteins are a family of five evolutionarily conserved “co‐E3s” that promote CRL activation via neddylation. Since DCNL proteins are required for CRL activation in vitro, we hypothesized that specific DCNLs might be localized to the distal nephron to downregulate WNK kinases via the KLHL3/CUL3 complex. A kidney RNAseq database (Lee JW JASN 2015) detected DCNL4 transcripts in microdissected distal nephron segments, particularly the connecting tubule and cortical collecting duct. In immunohistochemistry and immunofluorescence studies, DCNL4‐specific antibodies detected a strong cytoplasmic signal in distal nephron intercalated cells (ICs). Notably, the signal was absent in the distal convoluted tubule and loop of Henle, the classical sites of WNK pathway activity. Recently, we showed that the abundance of the “long” kinase active form of WNK1 (L‐WNK1) is increased in ICs during dietary potassium loading. Consistent with this finding, dietary potassium loading (5% K+ with mixed citrate chloride and carbonate x10d) increased the abundance of the L‐WNK1 effector kinase SPAK in ICs, and decreased the abundance of DCNL4 by 53% by immunoblotting. Conversely, 10d of dietary K+ restriction upregulated DCNL4 in ICs and reciprocally decreased the abundance of L‐WNK1. In 293 cells, dicer substrate RNA‐mediated knockdown of DCNL4 increased endogenous steady state L‐WNK1 abundance, while overexpression of DCNL4 in the presence of KLHL3 and CUL3 enhanced L‐WNK1 degradation. These data support a model in which DCNL4 functions as an IC‐specific brake that downregulates L‐WNK1 and SPAK activity during hypokalemia, a process that is released during dietary potassium loading to activate IC‐mediated K‐secretion, possibly via basolateral NKCC1 cotransporters and apical BK channels. Thus, these findings suggest a novel physiological role for a specific DCNL protein in the regulation of potassium homeostasis.Support or Funding InformationNIH R01DK098145, R01DK038470, P30DK079307This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.