Characterization of the kinase activity of a WNK4 protein complex

Abstract

Mutations in WNK4 protein kinase cause pseudohypoaldosteronism type II (PHAII), a genetic disorder that is characterized by renal NaCl and K(+) retention leading to hypertension and hyperkalemia. Consistent with this, WNK4 is known to regulate several renal tubule transporters, including the NaCl cotransporter, NCC, and the K(+) channel, ROMK, but the mechanisms are incompletely understood, and the role of the kinase activity in its actions is highly controversial. To assay WNK4 kinase activity, we have now succeeded in expressing and purifying full-length, enzymatically active WNK4 protein from HEK293 cells. We show that full-length wild-type WNK4 phosphorylates oxidative stress response kinase 1 (OSR1) and Ste20/SPS1-related proline/alanine-rich kinase (SPAK) in vitro. Introducing the PHAII-associated mutations, E559K, D561A, and Q562E, into our protein had no significant effect on this phosphorylation. We conclude that PHAII is unlikely to be caused by abnormal WNK4 kinase activity. We also made the intriguing observation that inactivating mutations of the WNK4 kinase domain did not completely abolish in vitro phosphorylation of OSR1/SPAK. Led by this, we identified a novel 40-kDa kinase that associates specifically with the COOH-terminal half of WNK4 and is able to phosphorylate both WNK4 and SPAK/OSR1. We suggest that this 40-kDa kinase functions in the WNK4 signal transduction pathway and may mediate some of the physiological actions attributed to WNK4.

Fig. 1.

Expression and in vitro kinase assay of full-length (FL) WNK4. A: wild-type (WT) and mutant WNK4 proteins, expressed and recovered from HEK293 cells with the high-salt and detergent (HSD) protocol, were separated by SDS-PAGE (0.5 μg of protein/lane) and the gel was silver-stained. As a negative control for WNK4 expression, a lysate from HEK293 cells transfected with vector alone was also treated with the HSD protocol and included in the SDS-PAGE (vector). B: WT and mutant WNK4 proteins (0.5 μg of protein/sample), purified with the HSD protocol, were incubated with kinase-inactive OSR1 (D164A) in the presence of γ³²P-ATP. In vitro phosphorylated OSR1 was visualized after SDS-PAGE by 1 h of autoradiography. To exclude OSR1 autophosphorylation, the mock protein preparation from cells transfected with vector alone was incubated with OSR1 in the presence of γ³²P-ATP (vector). C: OSR1 phosphorylation was quantified by scintillation counting of phosphorylated bands (both bands of OSR1 doublet), excised from the dried gel. OSR1 phosphorylation is plotted as the percent ³²P incorporation compared with WT (means ± SD, n = 3 independent experiments) and statistical significance was determined by repeated-measures ANOVA. PHAII, pseudohypoaldosteronism type II.

Fig. 2.

In-gel kinase assay of WNK4. A: WT WNK4, expressed and purified from HEK293 cells, was recovered by regular (Reg) or HSD tandem affinity purification (TAP) protocol, separated by SDS-PAGE, and visualized by silver staining. B: protein samples from A were separated on a polyacrylamide gel in which OSR1-D164A had been copolymerized. After renaturation, in-gel phosphorylation was carried out in the presence of γ³²P-ATP and phosphorylated bands were visualized by overnight autoradiography. C: WT and mutant WNK4, prepared by the HSD protocol, was assayed for in-gel phosphorylation of OSR1 as in A. A sample prep from cells transfected with vector alone was included as a negative control for OSR1 phosphorylation by nonspecific copurifying kinases (vector).

Fig. 3.

WNK4 expressed in different cell lines. A: Chinese hamster ovary cells (CHO) and African green monkey kidney cells (COS-7) were transfected as indicated with either FL or Δ593 truncated (Δ593) WNK4 constructs. Proteins were recovered by regular protocol and visualized on a silver-stained 7.5% polyacrylamide gel. B: in-gel phosphorylation with the indicated samples was carried out as described in Fig. 2. Phosphorylated bands were visualized by 3-h autoradiography.

Fig. 4.

Diagram of the WNK4 truncation constructs. The Δ symbol followed by a number indicates the amino acid in the WNK4 polypeptide sequence after which the COOH terminal of the protein was truncated. FL denotes FL protein. TAP, tandem affinity purification tag; AID?, predicted autoinhibitory domain; CC, coiled-coil; PHAII, stretch of acidic amino acids where PHAII-associated mutations are located; c-myc, myc epitope tag.

Fig. 5.

Expression and in-gel kinase assay of COOH-terminal truncated WNK4 proteins. A: FL and COOH-terminal truncated WNK4 proteins were expressed and recovered from HEK293 cells with the regular TAP protocol and then separated on a 7.5% polyacrylamide gel and visualized by silver staining. B: NH₂ terminal of the WNK4 proteins shown in A was detected by a streptavidin blot. C: COOH terminal of the WNK4 proteins shown in A detected by immunoblotting with anti-myc antibody. D: assay of in-gel phosphorylation of OSR1-D164A by FL and COOH-terminal truncated WNK4 proteins. ³²P-phosphorylated proteins were visualized by 3-h autoradiography.

Fig. 6.

Expression and in-gel kinase assay of NH₂-terminal truncated WNK4 protein. A: FL and the indicated truncated WNK4 proteins were expressed and recovered from HEK293 cells with the regular TAP protocol and then separated on a 7.5% polyacrylamide gel and visualized by silver staining. B: assay of in-gel phosphorylation of OSR1-D164A by the NH₂-terminal truncated WNK4 594–1222, compared with FL WNK4 and Δ593. ³²P-phosphorylated proteins were visualized by 3-h autoradiography.

Fig. 7.

SPAK is a substrate of both WNK4 and p40. In vitro in-solution kinase assays were performed with the indicated WNK4 proteins expressed and purified from HEK293 cells, using a GST fusion protein of kinase-inactive SPAK (D212A) as the substrate (predicted molecular mass, 88 kDa). Left: FL constructs of WT WNK4 and the indicated kinase-inactive or PHAII mutants. Right: FL WT WNK4 compared with a COOH-terminal fragment (594–1222) of WT WNK4 lacking the kinase domain.

Fig. 8.

Role of the SPAK/OSR1-binding domain, RFQV, in WNK4. In vitro in-solution kinase assays were performed with the following TAP-purified FL WNK4 proteins: WT, a mutant in which the SPAK/OSR1-binding site was inactivated (RAQV), and a kinase-inactive mutant (D318A), using as substrates either OSR1 (lanes 2–4) or SPAK (lanes 5–7). A: Coomassie blue-stained gel. B: autoradiograph.

Fig. 9.

Characterization of p40 activity by in-gel kinase assay. Either OSR1 (A) or SPAK (B) was copolymerized into the gel, through which FL WT, FL RAQV mutant, or the NH₂-terminal fragment of WNK4 (Δ593) was electrophoresed and assayed in-gel in the presence of ³²P.