We have recently shown that SPAK and OSR1, two Ste20p-like serine-threonine kinases, interact with cation-chloride cotransporters, including the Na-K-2Cl cotransporter, NKCC1 (45
). We later showed that WNK4, a kinase involved in pseudohypoaldosteronism (55
), also interacts with SPAK through a RFQV binding motif (44
) and that this interaction between the two kinases results in significant activation of NKCC1 under isosmotic conditions (18
). Because the catalytic activity of both kinases is required for NKCC1 activation and because, in contrast to SPAK, WNK4 failed to interact directly with the cotransporter, we proposed a hierarchy of events in which WNK4 activates SPAK, which in turn phosphorylates NKCC1 (18
). Consistent with this idea, Vitari and coworkers have recently presented direct evidence that WNK1 and WNK4 phosphorylate and activate SPAK and OSR1 (54
Activation of many protein kinases requires phosphorylation of the activation segment or loop between two highly conserved tripeptide motifs (DFG… .APE) (38
). Crystallographic models of phosphorylated and nonphosphorylated protein kinases have demonstrated large movements in their activation loop, allowing better substrate access to the active site, suggesting that the activation loop may function as a “door” to the substrate pocket (25
). On the basis of the high degree of conservation between SPAK and this particular group of protein kinases, we speculated that phosphorylation of threonine residues located in the activation loop might be critical in the activation of the kinase. Our experiments show that mutating residues T243 or T247 into alanines greatly impacted kinase activity, as determined through autophosphorylation and transphosphorylation of the N-terminal tail of NKCC1, whereas alanine substitution of T231 and T236 remained silent (Fig. ). In parallel to these in vitro phosphorylation experiments, we performed functional experiments with the threonine mutants in Xenopus laevis
oocytes and confirmed the requirement of T243 and T247 for NKCC1 activation (Fig. ).
Substrate recognition of many protein kinases depends, at least partly, on residues flanking the specific site of substrate phosphorylation (or “P site”) with residues N terminal to the P site numbered P−1, P−2, and P−3 and residues C terminal to the P sites numbered P+1, P+2, P+3, etc. (1
). In the case of the Ste20p-like protein kinase TAO2, it was shown that its physiological substrates MEK3 and MEK6 both possess hydrophobic residues immediately following the P site (P+1) in both of their two phosphorylation sites (58
). For NKCC1, one of the physiological substrates of SPAK, Darman and Forbush have identified the principal P site of shark NKCC1 as T189 (11
). Consistent with observations of MEK3 and MEK6, this site is directly followed by a hydrophobic methionine residue. Amino acid sequence alignment of the catalytic domain of mammalian Ste20p-like kinases reveals conservation of a quartet of hydrophobic residues forming a pocket that interacts with the P+1 site. As seen in Fig. , the hydrophobic residues lining the P+1 pocket in SPAK are F244, M251, P253, and L278. Further analysis of the activation loop of SPAK reveals that residues T243 and T247, newly defined as critical for SPAK activity, are themselves directly followed by hydrophobic residues, phenylalanine following T243 and a proline residue coming after T247, whereas T231 and T236 are followed by nonhydrophobic glycine and arginine residues, respectively. This observation suggests that T243 and T247 might be phosphorylated in a manner similar to the phosphorylation of Ste20p substrates. Interestingly, Vitari et al. identified T243 as a target of WNK1 and WNK4 (54
), and both kinases possess the conserved quartet of hydrophobic residues: V383, M390, P392, and M415 for mouse WNK1 and V333, M340, P342, and P365 for mouse WNK4. However, as our in vitro phosphorylation experiments showed that SPAK itself can incorporate phosphates on T243 and T247 (autophosphorylation), whether or not the P+1 pocket is involved is currently unknown. On the basis of the presence of a dimerization region in the regulatory domain of MST1, a Ste20p-like kinase involved in apoptosis, Glantschnig et al. (21
) argued that dimerization promotes phosphorylation between individual MST1 molecules. They indeed demonstrated intermolecular phosphorylation by using kinase-active and -inactive forms of MST1. Our yeast two-hybrid data indicate the absence of SPAK-SPAK interaction, making it therefore unlikely that SPAK possesses a dimerization domain. This observation is in agreement with the absence of any multimerization products on Western blots (44
). Because of the absence of SPAK-SPAK interaction, we thought it was important to ask whether, in the absence of dimerization, one could see intermolecular phosphorylation. Our experiments clearly indicate that autophosphorylation occurs intramolecularly, as the ability of SPAK to autophosphorylate appears dilution independent (Fig. ) and as active SPAK is unable to phosphorylate catalytically inactive SPAK (Fig. ).
An interesting observation was that Mn2+
, but not Mg2+
, was very effective as a cofactor in our in vitro phosphorylation experiments. This particular divalent metal ion specificity has been reported for a few kinases (2
) and nucleotide binding proteins (56
). Whether or not the same specificity exists in vivo remains to be determined. Slightly different conformational changes of the SPAK active site might be generated depending upon the nature of the divalent metal coordinated to the polyphosphate region of the nucleotide. Indeed, X-ray crystallography of CheA showed that the active site of this histidine kinase is greatly influenced by the divalent metal ion bound to the nucleotide, with Mg2+
enabling a more extensive conformational change than Mn2+
). Stronger autophosphorylation with Mn2+
in our in vitro experiments might indicate a more open or relaxed conformation of SPAK. In some cases, Mn2+
has also been shown to facilitate the use of GTP as a phosphoryl group donor (22
). This is clearly not the case with SPAK, where GTP cannot substitute for ATP (Fig. ). Since Mg2+
is more physiologically relevant, it is possible that in vivo, autophosphorylation of the threonines in the activation loop might be minimal, allowing for phosphorylation by other upstream kinases such as WNK1 and/or WNK4.
Experiments performed using internally dialyzed squid giant axons in the late 1970s revealed that Na-K-2Cl cotransport is inhibited by high intracellular Cl−
concentrations. This effect, reproduced in a variety of other cell types, is not related to the ion driving force, since increased intracellular Cl−
concentrations also inhibit Na-K-2Cl cotransport-mediated efflux. Of interest is the demonstration that NKCC1 phosphorylation increases with decreases in Cl−
concentrations (for a review, see reference 46
). We examined the Cl sensitivity of both SPAK and OSR1 phosphorylation and found an inhibitory effect in the physiological range (4 to 40 mM). Whether or not the effect of Cl−
concentrations on SPAK autophosphorylation is large enough to account for the exquisite sensitivity of NKCC1 to internal Cl−
concentrations in vivo remains to be determined.
Yeast two-hybrid experiments have previously demonstrated that OSR1, a kinase closely related to SPAK, interacts with the K-Cl cotransporter (KCC3) (44
). Immunofluorescence studies with polyclonal antibodies have also demonstrated colocalization of OSR1 and NKCC1 (35
). It is therefore not surprising that OSR1, like SPAK, when coexpressed with WNK4 activates NKCC1 in Xenopus laevis
oocytes. Consistent with these results, our in vitro phosphorylation experiments indicate that OSR1 exhibits many of the same kinetic properties as SPAK. Previous in vivo studies examining OSR1 phosphorylation have shown an increase in kinase activity when the cells were incubated with sorbitol (8
). This hyperosmotic activation of OSR1 is consistent with an increase in cotransporter activity under similar conditions, a process we showed to be at least partially related to SPAK (18
). Taken together, these results indicate that the shared homology between OSR1 and SPAK is sufficient for either kinase to serve as a modulator of NKCC1 activity.
Over the past 25 years, a variety of inhibitors and activators of both cotransporters have been identified. These pharmacological interventions generally produce opposite effects and therefore are thought to act on the kinases and/or phosphatases which regulate cotransporter activity. Here, we had the possibility of testing the direct effect of several of these agents on both SPAK autophosphorylation and substrate phosphorylation of NKCC1. Staurosporine is a potent inhibitor of the Na-K-2Cl cotransporter (17
) and an activator of the K-Cl cotransporter (5
). We report here that staurosporine inhibits SPAK activity by ~50% at drug concentrations between 0.1 and 1 μM, consistent with a measured 50% inhibitory concentration of 0.7 μM in avian erythrocytes (33
). K252a, another protein kinase inhibitor preventing Na-K-2Cl cotransport stimulation in a variety of cells (40
), also inhibited SPAK activity, although requiring slightly higher concentrations than staurosporine. In contrast, arsenite, which stimulates stress-activated protein kinases (27
) and MAPKs (32
) and markedly activates Na-K-2Cl cotransport in ferret red cells (17
), had little effect on SPAK autophosphorylation. This seems to indicate that the arsenite effect in ferret erythrocytes is not directly associated with the kinase that phosphorylates the cotransporter.
We also demonstrated a direct inhibitory effect of hydrogen peroxide on SPAK autophosphorylation as well as transphosphorylation of NKCC1. Hydrogen peroxide (H2
) has been shown to stimulate K-Cl cotransport (4
), although the oxidant was believed to act through a phosphatase rather than a volume-sensitive kinase, as calyculin substantially inhibited the H2
). Although there are no reports of a H2
effect on Na-K-2Cl cotransport, tert-butyl hydroperoxide has been shown to have an inhibitory effect on the “regulatory” kinase (15
). Thus, our data showing SPAK inhibition by H2
are consistent with oxidative reagents inhibiting K-Cl cotransport and activating Na-K-2Cl cotransport. K-Cl cotransport was first defined as a mechanism promoting volume-induced (14
) and NEM-induced (29
flux. First, it was proposed that the alkylating reagent reacts with thiol groups located on the transport molecule (for a review, see reference 28
). However, in light of the fact that NEM also inhibits Na-K-2Cl cotransport, consensus has moved toward the idea that NEM acts as a protein kinase inhibitor, antagonizing the effect of calyculin A (e.g., see reference 37
). The concentration of NEM (1 mM) required to affect K-Cl cotransport in native red cells (29
), rabbit KCC1 heterologously expressed in HEK293 cells (20
), and Na-K-2Cl cotransport in native red cells (37
) is relatively high. We showed that 1 mM NEM minimally affects SPAK autophosphorylation but significantly decreases the level of NKCC1 phosphorylation. This observation suggests that despite SPAK autophosphorylation of the activation loop threonine residues, NEM still prevents kinase-substrate interaction, cotransporter phosphorylation, and, ultimately, functional activation. Interestingly, the sulfhydryl oxidant, diamide, produced similar effects on SPAK autophosphorylation and NKCC1 transphosphorylation, further evidence that the effector sites of these agents lie between SPAK and the cotransporters.
The effect of NEM is intriguing, since it clearly delineates two separate events: SPAK autophosphorylation and kinase phosphorylation of the cotransporter. Data obtained with our deletion mutants (Fig. ) also demonstrate that kinase autophosphorylation by itself is not sufficient but that a portion of the regulatory domain proximal to the catalytic domain is necessary for substrate phosphorylation. The fact that WNK1 and WNK4 phosphorylate SPAK on residue S383 (located within this proximal region of the regulatory domain) indicates that access of the substrate to the kinase domain might depend on some conformation changes triggered by phosphorylation of the C terminus.
In summary, our results demonstrate that SPAK and OSR1 are kinases regulated through activation segment phosphorylation. We found that two of the four threonine residues (T243 and T247) within the activation loop were critical for SPAK autophosphorylation, and subsequent substrate phosphorylation and activation of NKCC1. Through mutagenesis studies, we determined that in vitro phosphorylation of these key threonine residues occurs by intramolecular autophosphorylation. We also found that although truncation of the regulatory domain allowed SPAK autophosphorylation, a proximal portion of the regulatory domain (~70 amino acids) was necessary for NKCC1 phosphorylation. Finally, we demonstrate that pharmacological inhibitors of NKCC1, i.e., staurosporine and K252a, directly affect SPAK autophosphorylation and substrate phosphorylation of the cotransporter. Taken together, these results clearly indicate that SPAK (and OSR1) likely represent two of the kinases which phosphorylate and activate NKCC1.