The recent discovery of mutations in the WNK1 and WNK4 genes that are responsible for a particular form of human hypertension (PHA2) has generated considerable interest in this new family of serine-threonine kinases and in the molecular mechanisms leading to high blood pressure (13
). The case of the ubiquitously expressed WNK1 gene is particularly intriguing. In two kindreds affected by PHA2, two overlapping large deletions in the first intron of the WNK1 gene were identified. These deletions have no effect on the gene product itself but instead modify WNK1 gene expression, as observed in the leukocytes of affected subjects (18
). In this study, we show that WNK1 gene expression is under the control of three alternative promoters, generating several WNK1 isoforms with a tissue-specific distribution and resulting in the production of isoforms with and without the entire kinase domain. Further variations are achieved by use of alternative splicing and alternative polyadenylation sites. A newly discovered exon is under the influence of an alternative promoter and a kidney-specific enhancer. The corresponding kidney-specific transcript has no serine-threonine kinase activity, shedding light on an unsuspected function of this protein.
Several proximal transcription start sites and alternative splicing events were identified for the human WNK1 gene. Exon 1 is surrounded by a large CpG island that spans 1.5 kb, and comparison of the sequence of this region with the human expressed sequence tag database revealed several potential transcription initiation sites (17
). Primer extension and 5′RACE-PCR experiments enabled us to identify two transcription start sites in the 5′ flanking region, 219 and 179 bp upstream from the first translation start codon. We also identified other transcription start sites within exon 1, 623 to 481 bp downstream from the first ATG codon, resulting in a WNK1 isoform with only part of the sequence encoded by exon 1, the first 639 nucleotides of the coding sequence having been eliminated. However, the predicted protein contains the entire kinase domain, with an ATG (codon 214) located four codons before the codon encoding the first amino acid of subdomain I of the kinase domain. There is no clear evidence that these two putative proteins have different functional roles, although a potential coiled-coil domain is predicted between residues 189 and 220. In that regard, it is interesting that various WNK1 subcellular localizations were recently observed among different epithelia (2
The two proximal transcription start sites were confirmed to be functional by the identification of two proximal promoters that were functionally active in vitro. A very high level of transcriptional activity was observed with the P1 [1200; −1] fragment in various cell lines, with only 20% of this activity obtained with the P1 [2500; −1] construct. Whether the presence of a repressor element within positions −2500 to −1200 can account for quantitative differences in expression in tissues such as heart, skeletal muscle, brain, and various epithelia remains to be tested. Primer extension experiments showed that the level of P2 promoter activity is higher in human kidney than in leukocytes. However, it should be noted that these experiments are very sensitive to structural conformations of mRNA and do not allow a relative estimation of each isoform. Indeed, they suggested that P2 was more dominant than P1 in kidney, whereas the QRT-PCR found that the P2-driven transcript accounts for only 18% of the longer kinase domain-containing transcripts.
Further variation in human WNK1 gene transcripts is achieved by the use of two polyadenylation sites and alternative splicing. Northern blot experiments with rat and human cells have demonstrated the presence of two main mRNA species of unknown molecular structure (18
). Both isoforms seem to be present in tissues in which WNK1 is produced, with the shorter of the two transcripts being produced mainly in the kidney. Our study demonstrates that the two ubiquitous isoforms, approximately 9 and 10.5 kb in length, are generated by the use of two different polyadenylation sites separated by 1.8 kb at the 3′ end of the human WNK1 mRNA. Poly(A) tails may affect the translation and stability of the mRNA (6
). WNK1 transcripts with the longer, 2.6-kb 3′ untranslated region are the most abundant. This is particularly striking in tissues in which the human WNK1 gene is highly expressed, such as kidney, skeletal muscle, heart, and brain.
The detection of WNK1 transcripts displaying alternative splicing of exons 9, 11, and 12, which encode nonidentical proteins, confirms previous reports (17
). These three exons are conserved between human, mouse, and rat. In rat, the first published WNK1 sequence lacked exons 11 and 12 (20
). Our RT-PCR analysis reveals that these exons are not frequently used, suggesting that they may have special roles only in small cell populations or during certain developmental stages. We also found a novel splicing site in intron 3, 83 bp upstream from exon 4. The corresponding isoform is produced in only small amounts and contains a premature stop codon, leading to a predicted protein of 394 residues that is truncated after catalytic kinase subdomain VII, which should therefore be inactive. Such rare alternative splicing events, leading to the introduction of a premature stop codon and the production of a noncatalytic enzyme have been described for the human α-galactosidase A gene, which is responsible for Fabry disease (7
). Abnormal regulation of the production of this transcript by mutation within an intron leads to a particular cardiac phenotype of Fabry disease. Preliminary data indicate that production of this truncated human WNK1 isoform is not affected in the leukocytes of affected individuals from our PHA2 kindred with deletions in intron 1 (data not shown).
The existence of two proximal promoters and alternative splicing events cannot account for the molecular structure of the kidney-specific WNK1 transcript. We demonstrate in this study that the renal isoform, which in length is undistinguishable from the short 9-kb ubiquitous isoform, is kidney specific. It has the long, 2.6-kb 3′ untranslated region but lacks the kinase subdomains encoded by the 5′ part of the gene. Another novel alternative promoter within intron 4 mediates the specific production of this variant. We identified a single transcription start site within exon 4a and thus the structure of this exon, which is present in human, mouse, and rat, with high levels of similarity over the 90 bp of the coding sequence. The predicted human renal WNK1 protein sequence lacks the first 437 residues of the kinase-active WNK1 isoform but contains a novel cysteine-rich region at the N terminus. We characterized the minimal promoter sequence of this transcript, since the region immediately upstream from exon 4a has transcriptional activity in vitro, in both renal and nonrenal cell lines, and we discovered an enhancer element far upstream from the transcription start site (nucleotide −4284 to −3447) that conferred a ~20-fold activation selectively in renal MDCK cells. Preliminary in situ hybridization experiments with embryonic mouse kidney indicate a late expression of this renal isoform that may explain why it is not expressed in human embryonic kidney (HEK 293) cells and thus why the enhancer had no effect in these cells (not shown). The enhancer region was restricted to a 157-bp fragment that contains sequences highly homologous to 5′ flanking sequences of the human NCC and kallikrein genes. Both share a similar expression profile restricted to the distal convoluted tubule. These results suggest that the renal-specific production of the kinase-deficient WNK1 isoform is due to a specific regulatory region mediating high levels of transcription in the distal tubule. Isolation of the transcription factors involved in the regulation of this enhancer might provide important new insights into the molecular events responsible for the specific expression of this isoform in the distal convoluted tubule and into the understanding of the physiopathological mechanism of the human WNK1 mutations responsible for PHA2.
The renal WNK1 isoform is the first reported isoform of the WNK family to be generated by the use of an alternative promoter and directly translated in a form predicted to be devoid of serine-threonine kinase activity. The existence of noncatalytic isoforms of other members of the kinase superfamily has been reported. A particular analogy can be made with the TrkB gene, which also spans a large region and generates a large number of different isoforms from alternative promoters, splicing sites, and polyadenylation sites (15
). In this gene, a specific brain isoform, devoid of the tyrosine kinase domain, is generated by the use of an alternative exon 19 (8
). As for this gene, the WNK1 kinase-defective isoform is conserved in humans and rodents, highlighting its probable physiological importance. Within the kidney, this truncated isoform is produced in very large amounts, about 10 times larger than those for the ubiquitous isoforms, in analyses of total mouse kidney extracts. In situ hybridization showed even more striking features, with exclusive, high-level production of this transcript in the distal convoluted tubule but only very weak and diffuse staining for the kinase domain-containing isoforms.
Currently, we can only speculate on the function and mechanism of action of the various WNK1 isoforms. It has been demonstrated in vitro that the complete rat protein is capable of autophosphorylation (20
), but no specific substrate has yet been identified. Similarly, the regulators of WNK1 expression and the signal transduction pathways activated by this protein remain to be determined. However, we know that changes in NaCl concentration may result in the activation of WNK1 in vitro (20
). Furthermore, the ubiquitous presence of this protein in several epithelia, such as pancreatic ducts, epididymis, sweat ducts, and colonic crypts, is consistent with a role for WNK1 in the regulation of sodium and chloride reabsorption (2
). However, this function cannot account for the strong expression of the full-length WNK1 isoforms in tissues such as heart and skeletal muscle, as observed in Northern blots with various species. It is possible that the WNK1 produced in tissues not involved in ion transport (heart and skeletal muscle) has another, as-yet-unknown, function. Rat WNK1 was the first mammalian member of this kinase family to be cloned and characterized (20
), and 18 WNK-like kinases in seven species have been identified from in silico alignment, with high levels of similarity in the kinase motif (17
). Several lines of evidence suggest that WNK kinases may play a role in signaling related to cell contact or adhesion: (i) WNK1 is present exclusively in multicellular organisms (17
), (ii) WNK4 is present in the tight junctions of the renal distal tubule (18
), (iii) in vitro studies suggest that WNK1 undergoes oligomerization (21
), and (iv) partial WNK1 clones have been isolated from prostatic carcinoma and colorectal cell lines (11
). Further investigation is required to determine whether the two proximal promoters and the alternative splices described here can account for the differences in WNK1 gene expression, protein structure, and distribution.
The renal isoform lacks most of the N-terminal kinase domain but does contain, in addition to the two coiled-coil domains and the PXXP motifs, an autoinhibitory domain recently described by Xu et al. (21
). This domain is conserved among the members of the WNK family and blocks the activity of the WNK1 kinase domain (21
). In many protein kinases, this domain is used to regulate kinase activity, by a mechanism involving its removal from the inhibitory site by activating signals (9
). WNK1 autoinhibition could be blocked, at least partially, by the formation of oligomers via the coiled-coil domains (21
). It therefore seems likely that, as for the TrkB long and short isoforms (15
), the full-length WNK1 isoform can bind the short renal isoform but not phosphorylate it, due to the absence of the critical serine residues. The renal kinase-defective WNK1 could therefore act as a dominant negative form of the catalytic WNK1 isoform. Alternatively, the marked predominance of the kinase-defective isoform within the distal convoluted tubule may reflect this isoform having a specific function in the kidney, regulating other kinases such as WNK4. The recent demonstration of WNK1 inhibition of the effect of WNK4 to reduce NCC-mediated Na uptake in Xenopus
oocytes suggests that the long WNK1 isoform may bind WNK4 within the cell, preventing its phosphorylation and/or migration to the membrane, thereby playing the role of a negative regulator (13
). If this is also true for the kidney-specific kinase-deficient isoform, interactions with WNK4, and possibly with other proteins, would then result in physiological regulation of the signaling pathways controlling the ion permeability of nephron epithelia.
Human WNK1 mutations causing PHA2 have been shown to be associated with overproduction of the short WNK1 transcript in the leukocytes of affected subjects. Therefore, they might increase NCC activity by inhibiting the negative physiological effects of WNK4 on the NCC leading to sodium retention and hypertension. This interaction model may account for human PHA2-causing mutations of the WNK1 and WNK4 genes being undistinguishable in terms of biological and clinical features. Analysis in vitro and in vivo of the consequences of the PHA2-causing deletions of intron 1 for WNK1 gene expression and protein structure should benefit from the results of this study, making it possible to obtain further insight into the molecular mechanisms of this particular form of human hypertension.