The WNK2 expression profile is unique among the WNK kinases. First, while WNK1, WNK3, and WNK4 are highly expressed in multiple Cl−
-transporting epithelia (19
), and most notably the nephron (16
), WNK2 is almost exclusively expressed in the brain, with no detectable expression in kidney. Second, in the CNS, while WNK1 is expressed throughout postnatal development, WNK3 is not significantly expressed until postnatal day 21 (19
) and WNK2 is expressed since the embryonic life. Third, within the brain, WNK1 is predominantly expressed in non-neuronal cells and WNK3 localizes to both neurons and glia (19
), but WNK2 is expressed primarily in cortical and thalamic neurons. Thus, WNK2 is more highly expressed in neurons and during early development than other WNKs. These differences might have important functional ramifications for the regulation of neuronal CCCs. In many other tissues in which WNKS and CCCs have been shown to co localize evidences show that WNKs are playing an important role in their regulation. In this regard, future development of WNK2 knock-out or knockin mice will be useful to analyze with detail the role that WNK2 might have on different parts of the brain function, during embryonic and in postnatal life.
It not clear at the moment how changes in osmolarity and/or intracellular chloride concentration modulates the activity of WNKs, but it has been observed that hypertonicity is clearly associated with activation of WNK1 by inducing autophosphorylation of the T-loop serine 328, known to be associated with activation of the kinase (37
). However, similar to what we have observed for WNK3 (17
) WNK2 bypasses normal tonicity requirements to activate NKCC1 and inhibit KCC2 in the Xenopus
oocytes. While caution must be exercised in extrapolating results to mammalian systems, oocytes have proven useful for studying the molecular mechanisms of CCC function and regulation (48
). The opposing effects of WNK2 on cellular Cl−
influx and efflux pathways would be expected to achieve a net increase in [Cl−
, a property previously described for WNK3 (17
). However, in contrast to WNK3, WNK2 is expressed during early development. As depicted in , after birth, the functional expression of the chloride-importing NKCC1 and chloride-exporting KCC2 cation-chloride cotransporters (CCC) is reciprocally related: NKCC1 is decreased, and KCC2 is increased. This shift is thought to underlie the polarity change in GABAergic signaling from excitatory in the immature brain to inhibitory in the mature brain. In the developing brain, WNK3 expression is low. In contrast, WNK2 is strongly expressed in both the developing and mature brain. The ontogeny of WNK2 expression, coupled with its ability to load Cl−
into cells, suggests WNK2 might be important for establishing the elevated [Cl−
]i seen early in development that is critical for the trophic functions of GABA excitatory signaling (2
). In adulthood, both WNK2 and WNK3 are highly expressed. An appropriate mixture of WNK kinases might confer certain neuronal groups, like the suprachiasmatic nucleus, with the ability to rapidly change [Cl−
, allowing for dynamic shifts in the response to GABA ().
Relationship between GABA excitatory and inhibitory effect and the expression of WNK2, WNK3, NKCC1, and KCC2 in the CNS from embryonic to postnatal life.
Regulation of [Cl−
by WNK2 might also be important for the role of the CCCs during regulatory volume increase (RVI) and regulatory volume decrease (RVD), key homeostatic counter-responses to maintain cell volume during shifts in extracellular osmolarity or intracellular solute content (). Acute activation of NKCC1 and other ion transport mechanisms (such as NHE1 functioning in parallel with a Cl−
exchanger), are important in RVI and operate in neurons (49
). KCCs like KCC3 are important for cell volume regulation via RVD in the nervous system (52
). The WNK2 ability to activate NKCC1 and inhibit the KCCs suggests it may participate in RVI in neurons. Additional experiments will be required to investigate whether WNK2 activity is affected by changes in cell volume, or is altered in pathologic states like cerebral edema.
WNK kinases exhibit kinase-dependent and -independent effects on different ion transport pathways. WNK2 regulation of the CCCs is likely dependent on its catalytic activity, since its activation of NKCC1 or inhibition of KCCs is lost by an inactivating mutation in its kinase domain. Since serine-threonine phosphorylation is the primary mode of CCC regulation, we presume WNK2 activates NKCC1 and inhibits KCC2 by promoting net cotransporter phosphorylation. This could be due to: 1) direct phosphorylation of the CCCs by WNK2; 2) indirect phosphorylation via another downstream kinase; or 3) inhibition of protein phosphatases. The fact that WNK2 forms a complex with SPAK in vivo
, that in this complex SPAK is phosphorylated at a consensus WNK-phospho site, and that SPAK has been shown to directly phsophorylate NKCC1 (35
), suggest WNK2 might activate NKCC1 indirectly via SPAK kinase. If so, this mechanism would be analogous to the WNK-SPAK-NCC pathway in kidney (46
). We and others have previously shown that WNKs modulation of CCCs is associated with changes in protein expression at the cell surface (17
). Here we shown that increased activity of NKCC1 induced by WNK2 or WNK3 is due to both, an increase in protein levels and also an increase in the surface expression of the cotransproter. In this regard, it has been shown that decreased activity of NCC induced by WNK4 is due to both, a reduction in the surface expression of the cotransporter (31
) and to a increased degradation of the NCC via lysosomes (57
). In contrast, the down-regulation of KCC4 activity by WNK2 or WNK3 is more dependent on their effect upon the surface expression of the cotransporter.
WNK2 might utilize a similar mechanism to inhibit KCC2; however, SPAK does not appear to phosphorylate KCC2, and recent experiments demonstrate that WNKs and SPAK/OSR1 are essential for KCC phosphorylation (28
). Because the regulation of KCCs by catalytically inactive WNK3 is prevented with protein phosphatase inhibitors such as calyculin or cyclosporin A, it is possible that a protein phosphatases are involved in the WNK2-KCC mechanism (18
SPAK and oxidative-stress response 1 (OSR1) kinase have been shown to bind, phosphorylate, and directly activate NKCC1 in multiple cell systems, including dorsal root ganglion neurons (14
). In this study, we isolated WNK2 in a phosphoprotein complex with SPAK from native mouse brain, suggesting these two kinases might work in concert to regulate neuronal CCCs. WNK1, WNK3, and WNK4 are known to interact with OSR1 and SPAK, and WNK1 and WNK4 phosphorylate Thr-233 and Ser-373 in SPAK, and Thr-185 and Ser-325 in OSR1 (35
). SPAK activity requires phosphorylation at Thr-233 and Ser-373 (59
). In our study, we have shown that SPAK is phosphorylated at Ser-366 and Ser-383 when complexed to WNK2 in vivo
. These results suggest that a WNK2-SPAK-CCC pathway is operative in the brain. Further experiments will be required to examine the functional role of this pathway in both the CNS, including the spinal cord, and the PNS (e.g.
in dorsal root ganglion cells).
WNK2 can be phosphorylated in at least 12 different sites. From the sequence flanking these phosphorylation sites, the Networkin prediction program (60
) identified potential kinases that might phosphorylate WNK2. These include sites for Rho-associated protein kinase 1 (DMPK), cAMP-dependent protein kinase (PKA), casein kinase II (CKII), PKC protein kinase CΔ (PKC), and cyclin-dependent kinase 5 (CDK5) (). Among these putative upstream kinases, P38MAPK, PKA, and Cdk5 are particularly interesting owing to prior evidence of their role in neuronal function. Because WNK kinase regulation might be ultimately determined by the N- and C-terminal regions flanking the kinase domain, any of these kinases are candidates for WNK co-regulatory kinases.
At present, it is unclear if WNK2's regulation of CCCs is linked to its role in cell growth and proliferation (61
). Hong et al.
) recently identified WNK2
as a tumor suppressor gene in a large-scale genomic and epigenomic analysis of human infiltrative gliomas. Epigenetic silencing of WNK2 has also been shown in all grades of meningioma (23
). Point mutations in other WNKs
have also been associated with breast, lung and colonic cancer (62
). In this context, it is interesting that NKCC1 has recently been shown to be the major pathway for Cl−
accumulation in glioma cells, and genetic or pharmacologic inhibition of NKCC1 is associated with a marked reduction in glioma cell invasion in vivo
). These issues will be topics of future experiments.