Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling

Abstract

Significance Calcineurin inhibitors (CNIs) are potent immunosuppressants; hypertension and hyperkalemia are common adverse effects. Activation of the renal Na-Cl cotransporter (NCC) is implicated in this toxicity; however, the mechanism is unknown. CNIs’ renal effects mimic the hypertension and hyperkalemia resulting from mutations in WNK kinases or in KLHL3-CUL3 ubiquitin ligase. WNKs activate NCC and are degraded by ubiquitylation upon their binding to KLHL3. The binding of WNKs to KLHL3 is prevented by KLHL3 mutations or by PKC-mediated KLHL3 phosphorylation at serine 433. This work shows that calcineurin dephosphorylates KLHL3S433, promoting WNK4 degradation. Conversely, CNIs inhibit KLHL3S433 dephosphorylation, preventing WNK degradation. These findings implicate calcineurin in the normal regulation of KLHL3’s binding of WNK4 and identify a direct target causing CNI-induced pathology. 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 (KLHL3S433-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.