1. Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling.
- Author
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Ishizawa K, Wang Q, Li J, Yamazaki O, Tamura Y, Fujigaki Y, Uchida S, Lifton RP, and Shibata S
- Subjects
- Adaptor Proteins, Signal Transducing, Angiotensin II genetics, Angiotensin II metabolism, Animals, Calcineurin genetics, Calcineurin Inhibitors administration & dosage, Cullin Proteins genetics, Gene Expression Regulation drug effects, Germ-Line Mutation genetics, Humans, Hyperkalemia genetics, Hyperkalemia metabolism, Hyperkalemia pathology, Hypertension metabolism, Hypertension pathology, Kidney drug effects, Kidney metabolism, Kidney Tubules, Distal metabolism, Kidney Tubules, Distal pathology, Mice, Microfilament Proteins, Multiprotein Complexes genetics, Phosphorylation, Renal Insufficiency chemically induced, Renal Insufficiency drug therapy, Renal Insufficiency pathology, T-Lymphocytes drug effects, T-Lymphocytes metabolism, Tacrolimus toxicity, Ubiquitination, Carrier Proteins genetics, Hypertension genetics, Protein Serine-Threonine Kinases genetics, Renal Insufficiency genetics
- Abstract
Calcineurin is a calcium/calmodulin-regulated phosphatase known for its role in activation of T cells following engagement of the T cell receptor. Calcineurin inhibitors (CNIs) are widely used as immunosuppressive agents; common adverse effects of CNIs are hypertension and hyperkalemia. While previous studies have implicated activation of the Na-Cl cotransporter (NCC) in the renal distal convoluted tubule (DCT) in this toxicity, the molecular mechanism of this effect is unknown. The renal effects of CNIs mimic the hypertension and hyperkalemia that result from germ-line mutations in with-no-lysine (WNK) kinases and the Kelch-like 3 (KLHL3)-CUL3 ubiquitin ligase complex. WNK4 is an activator of NCC and is degraded by binding to KLHL3 followed by WNK4's ubiquitylation and proteasomal degradation. This binding is prevented by phosphorylation of KLHL3 at serine 433 (KLHL3
S433-P ) via protein kinase C, resulting in increased WNK4 levels and increased NCC activity. Mechanisms mediating KLHL3S433-P dephosphorylation have heretofore been unknown. We now demonstrate that calcineurin expressed in DCT is a potent KLHL3S433-P phosphatase. In mammalian cells, the calcium ionophore ionomycin, a calcineurin activator, reduces KLHL3S433-P levels, and this effect is reversed by the calcineurin inhibitor tacrolimus and by siRNA-mediated knockdown of calcineurin. In vivo, tacrolimus increases levels of KLHL3S433-P , resulting in increased levels of WNK4, phosphorylated SPAK, and NCC. Moreover, tacrolimus attenuates KLHL3-mediated WNK4 ubiquitylation and degradation, while this effect is absent in KLHL3 with S433A substitution. Additionally, increased extracellular K+ induced calcineurin-dependent dephosphorylation of KLHL3S433-P These findings demonstrate that KLHL3S433-P is a calcineurin substrate and implicate increased KLHL3 phosphorylation in tacrolimus-induced pathologies., Competing Interests: Conflict of interest statement: R.P.L. is a nonexecutive director of Roche and its subsidiary Genentech., (Copyright © 2019 the Author(s). Published by PNAS.)- Published
- 2019
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