Characterization of the Interaction of the Stress Kinase SPAK with the Na+-K+-2Cl– Cotransporter in the Nervous System

Abstract

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related prolinealanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K ؉ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activitydependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress. Cation-chloride cotransporters, which mediate the tightly coupled, electroneutral movement of cations (Na ϩ and K ϩ ) together with Cl Ϫ , can be divided into Na ϩ -dependent transporters, such as Na-K-2Cl cotransporters (NKCC1-2) and the Na-Cl cotransporter (NCC), and Na ϩ -independent K-Cl cotransporters (KCC1-4). All of these transporters have a well conserved topology with large intracellular amino-terminal and carboxyl-terminal tails and 12 transmembrane spanning domains. The core protein shares some homology to amino acid permeases (for reviews see Refs. 1 and 2). A variety of stimuli regulates these transporters, including hormonal (3), cytokines (4, 5), cell volume (6, 7), oxidative stress (8), etc. There is also accumulating evidence that cation-chloride cotransporters participate in pathways leading to cell differentiation, growth and proliferation (9, 10), and apoptosis (11,12). At the molecular level, the activation-deactivation of these cotransporters mostly involves phosphorylation/dephosphorylation mechanisms, the details of which are still the subject of intense investigation.To identify proteins that directly interact and regulate cation-chloride cotransporters, we recently performed a yeast twohybrid screen using the cytosolic amino-terminal tail of KCC3. We identified two closely related kinases that bind to KCC3, NKCC1 and NKCC2 (13). These kinases belong to the group of Ste20 kinases that function as regulators of MAPK 1 cascades (14). The first kinase, SPAK (Ste-20 related proline-alaninerich kinase, or PASK, as the rat homologue), is highly expressed in epithelia and neurons (15). Its gene is located on human chromosome...

WNK3 modulates transport of Cl - in and out of cells: Implications for control of cell volume and neuronal excitability

“…Because hypertonicity can lead to robust phosphorylation of NKCC1 in the absence of exogenous WNK3 in the oocyte system (see Fig. 4), and because of evidence that the kinase PASK may be responsible for the direct phosphorylation of regulatory sites of NKCC1 (44,45), we presume that WNK3 is not directly phosphorylating its targets but instead is regulating downstream kinases͞phosphatases that ultimately act at the target. WNK3's localization to intercellular junctions, distant from the apical sites of these transporters, is consistent with this speculation.…”

WNK lies upstream of kinases involved in regulation of ion transporters

“…To investigate the effect of SPAK and OSR1 phosphorylation by WNKs, the authors analysed the effect of WNKs and SPAK or OSR1 on phosphorylation of the N-terminal domain of NKCCl. This was a very important experiment, because it is well known that the N-terminal domain of this co-transporter contains two threonine residues that are phosphorylated during its activation [11], and that two SPAKbinding motifs are located upstream of these threonine residues [10], in addition to experimental data suggesting that SPAK regulates NKCCl activity [10,12]. The authors observed that SPAK or OSR1 induced phosphorylation of the NKCCl Nterminal domain, but only when co-incubated with WNK1 or WNK4.…”

“…Four protein bands were detected in the WNK1 immunoprecipitate that were not seen in the control: two corresponded to WNK1 and WNK3, which was not a surprise since it is known that WNKs interact with each other [6]; a third band was the SDB84 antigen, but direct interaction between WNK1 and SDB84 was not confirmed; and the fourth band was a member of the STE20 family of kinases known as SPAK (STE20/SPS1-related proline/alanine-rich kinases). This last observation was pursued since it has been shown that SPAK, and the related OSR1 (oxidative stress response kinase-1), are involved in regulation of the basolateral isoform of NKCCl, and that WNK4 and SPAK can interact in a yeast two-hybrid system [9,10]. Of the four members of the STE20 family, the authors observed that WNK1 and WNK4 are able to physically interact with SPAK and OSR1, but not with the pseudokinases STRADα (STE20 related-adaptor α) and STRADβ.…”

“…The study of Vitari et al [8] is purely biochemical; however, a concomitant study provides the physiological counterpart of their findings. Gagnon et al [14], following their previous observations that SPAK interacts with NKCCl and that SPAK and WNK4 can interact in a yeast two-hybrid system [9,10], studied the effects of SPAK and WNK4 together on the activity of NKCCl. Gagnon et al [14] observed that co-injection of Xenopus laevis oocytes with NKCCl, SPAK and WNK4 cRNA resulted in a significant increase in NKCCl activity and insensitivity to external osmolarity or cell volume.…”

WNK1 and OSR1 regulate the Na + , K + , 2Cl − cotransporter in HeLa cells

“…Residues in the N-terminal cytoplasmic domain of NKCC1 are phosphorylated and increase its activity (10, 11). Although there is disagreement regarding the regulatory action of SPAK, one group of studies showed that overexpression of inactive SPAK blocked NKCC1 phosphorylation and activation (8,12). Certain sites phosphorylated in NKCC1 are conserved in NKCC2 and the sodium chloride cotransporter, another SLC12 family member, and are known to be phosphorylated in NKCC2 in response to vasopressin (13).…”