ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: SA-PO1056

Characterization of Kidney-Specific, C-Terminally Truncated Forms of WNK4 Kinase

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Authors

  • Murillo-de-Ozores, Adrian R., INCMNSZ, CDMX, Mexico
  • Bazua-Valenti, Silvana, INCMNSZ, CDMX, Mexico
  • Rodriguez-Gama, Alejandro, INCMNSZ, CDMX, Mexico
  • Leyva-Rios, Karla, INCMNSZ, CDMX, Mexico
  • Vázquez, Norma Hilda, INCMNSZ, CDMX, Mexico
  • Yang, Chao-Ling, Oregon Health & Science University, Portland, Oregon, United States
  • Loffing, Johannes, Institute of Anatomy, UZH, Zurich, Switzerland
  • Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
  • Gamba, Gerardo, INCMNSZ, CDMX, Mexico
  • Castañeda-Bueno, Maria, INCMNSZ, CDMX, Mexico
Background

The kinase WNK4 is an important regulator of renal salt handling. Mutations in this gene cause Familial Hyperkalemic Hypertension, mainly due to overactivation of the renal NaCl cotransporter, NCC. In addition to the full-length WNK4, we have observed shorter forms of this kinase in kidney lysates.

Methods

Western Blot, LC-MS/MS, immunoprecipitation, site-directed mutagenesis, transfection in HEK293 cells, and in vitro proteolytic assays were performed to characterize the short forms of WNK4.

Results

In Western blot assays, using WNK4-/- mice as control and two different N-terminal WNK4 antibodies, we observed lower bands (between 130 and 95 kDa) corresponding to WNK4 fragments in renal lysates of WNK4+/+ mice. These bands were not observed in other tissue lysates. LC-MS/MS confirmed that these bands correspond to WNK4 fragments that lack a C-terminal segment. One of these WNK4 forms may be produced by proteolytic cleavage, as we found that recombinant WNK4 is cleaved when incubated with kidney lysate. This process was prevented by a Zn2+ chelator. In HEK293 cells, we observed that truncation of WNK4’s C-terminus at several positions increases kinase’s activity towards SPAK, unless the truncated segment is large enough to include the SPAK binding site. This gain of function is caused by the loss of a protein phosphatase 1 (PP1) binding site. Cotransfection of PP1 causes dephosphorylation of WNK4, while this effect is abrogated in the WNK4-PP1 binding site mutant. Biochemical evidence suggests that the WNK4 short forms detected in vivo may lack the SPAK binding site and thus may not behave as constitutively active kinases.

Conclusion

We show the identification of short, C-terminally truncated, and kidney-specific WNK4 forms, at least one of which may be product of proteolysis. Moreover, this work allowed us to identify a bona fide PP1 binding site in the C-terminal region of WNK4 that modulates its activity towards SPAK-NCC.

Funding

  • Government Support - Non-U.S.