Characterization of SPAK and OSR1, Regulatory Kinases of the Na-K-2Cl Cotransporter

Abstract

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn 2؉ rather than Mg 2؉ . We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.Cation-chloride cotransporters (e.g., Na-K-2Cl and K-Cl cotransporters) serve multiple fundamental functions in a wide variety of tissues and organs. These include influencing ion and fluid movements in secreting or reabsorbing epithelia, control of CNS excitability, and cell volume regulation, proliferation, and survival (for a review, see reference 19). On the basis of the observations that the cotransporters are phosphoproteins and that phosphatase inhibitors affect cotransport activity, it is generally agreed that cotransporter regulation is mainly mediated through phosphorylation-dephosphorylation mechanisms. Activity of NKCC1, for instance, correlates directly with the phosphorylation state of the protein (33). For the past 20 years, the identity of the kinase in question has remained elusive. We have recently shown direct interaction between cation-chloride cotransporter and two Ste20p-related serine/threonine kinases, SPAK and OSR1 (45).There are some 30 protein kinases identified in mammals related to the budding Saccharomyces cerevisiae sterile 20 protein kinase (Ste20p). In 1991, Dan et al. divided the mammalian Ste20p-like kinases into two subgroups: the p21-activated kinases (PAKs; two subfamilies), which are characterized by a carboxyl-terminal catalytic domain, and the germinal center kinases (GCKs; eight subfamilies), which have their catalytic domain located at the amino terminus (10). Most Ste20p-like kinases stimulate mitogen-activated protein kinase (MAPK) cascades and thus participate in the modulation of cell motility (7), cell growth (31), and apoptosis (23).The GCK6 subfamily comprises two kinases: SPAK/PASK (Ste...

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

“…The generation of SPAK knock-out (KO, SPAK Ϫ/Ϫ ) mice has been described in detail previously (11,24). Briefly, the SPAK gene (Stk39) was disrupted by duplicating exon 6 and inserting tyrosinase (Tyr), neomycin resistance (neo), and 50 hypoxanthine phosphoribosyltransferase (hprt) genes between the two exons.…”

“…The day after isolation, oocytes were injected with 50 nl of water containing 15 ng of mouse NKCC1 cRNA. The third day, oocytes were injected with 50 nl of water containing 10 ng of each kinase cRNA (11). K ϩ uptakes were measured on day 5 by using 5 mCi/ml of 86 Rb, as tracer.…”

Protein kinase WNK3 increases cell survival in a caspase-3-dependent pathway

“…WNK4 is expressed in the distal nephron where it appears to regulate paracellular chloride (Cl À ) permeability as well as the traffic of epithelial ion channels such as the sodium-chloride (Na þ -Cl À ) cotransporter and the renal potassium channel ROMK (reviewed in Kahle et al, 2004a;Gamba, 2005). WNK1 has been found to act as an upstream activator of the extracellular signal-regulated kinase (ERK)5 pathway , to phosphorylate synaptotagmins-2, a protein involved in calciumdependent membrane fusion during exocytosis and endocytosis and to activate the protein kinases SGK1 and SPAK or OSR1 (Moriguchi et al, 2005;Vitari et al, 2005;Gagnon et al, 2006), which regulate activation of various epithelial ion channels. WNK1 itself is a substrate for protein kinase B upon activation of phosphatidyl inositol-3-kinase (Vitari et al, 2004;Jiang et al, 2005).…”

Pathogenesis of Inflammatory Bowel Diseases

“…The diversity of domains present in SPAK protein might be associated with a variety of biological roles. For example, SPAK has been shown to play roles in cell differentiation, cell transformation and proliferation, and regulation of chloride transport (Piechotta et al, 2002;Gagnon et al, 2006). More importantly, a linkage has been established between SPAK and inflammation, SPAK, as an upstream kinase to Na + -K + -2Cl − co-transporter 1 (NKCC1), can phosphorylate Thr203, Thr207, and Thr212 amino acids on NKCC1, which play an important role in inflammation (Topper et al, 1997).…”