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

Abstract

Oxidative stress-responsive kinase (OSR) 1 and sterile20-related, proline-, alanine-rich kinase (SPAK) are Ste20p-related protein kinases that bind to the sodium, potassium, two chloride cotransporter, NKCC. Here we present evidence that the protein kinase with no lysine [K] (WNK) 1 regulates OSR1, SPAK, and NKCC activities. OSR1 exists in a complex with WNK1 in cells, is activated by recombinant WNK1 in vitro, and is phosphorylated in a WNK1-dependent manner in cells. Depletion of WNK1 from HeLa cells by using small interfering RNA reduces OSR1 kinase activity. In addition, depletion of either WNK1 or OSR1 reduces NKCC activity, indicating that WNK1 and OSR1 are both required for NKCC function. OSR1 and SPAK are likely links between WNK1 and NKCC in a pathway that contributes to volume regulation and blood pressure homeostasis in mammals.blood pressure ͉ kinase ͉ osmotic ͉ stress P rotein kinase cascades mediate cellular responses to extracellular signals and environmental change. A large group of protein kinases with remarkably complex and diverse functions are related to the yeast protein kinase Ste20p (1). Ste20p was identified as a component of a mitogen-activated protein kinase cascade in the yeast pheromone-induced mating pathway (2, 3). One substrate of Ste20p is Ste11p, the MAP kinase kinase kinase in the module; thus, Ste20p is the prototypical MAP4K. Ste20p also controls cytoskeletal reorganization required for budding in yeast. Ste20p kinases coordinate the activities of downstream molecules not only because of their catalytic activities but also because of their capacities to bind and organize upstream molecules, including receptors and adaptors, and downstream molecules, including cytoskeletal elements and other protein kinases.More than 30 Ste20p-related kinases are encoded in the human genome in two structurally distinct subfamilies, the p21-activated kinase (PAK) subfamily and the germinal center kinase (GCK) subfamily (1). The PAKs bind to GTP-liganded forms of the Rho family small G proteins Rac and Cdc42 and are key regulators of cytoskeletal organization and cell motility (4). The germinal center kinase-related kinases all contain N-terminal catalytic domains and diverse C-terminal domains that were used to categorize them into eight subfamilies (1).OSR1 and the sterile20-related, proline-, alanine-rich kinase (SPAK) are the only two mammalian protein kinases in the germinal center kinase-VI subfamily. Although it has not been shown to be sensitive to oxidative stress, OSR1 was named for its similarity to oxidative stress-responsive kinase SOK1 (Ste20͞ oxidant stress response kinase-1) (5). OSR1 does respond to osmotic stress and phosphorylates PAK1, inhibiting its responsiveness to Cdc42 (6). In addition to their similar kinase domains, OSR1 and SPAK share two conserved C-terminal regions, known as PF1 and PF2 domains. The PF1 domain lies immediately C-terminal to the protein kinase domain and is required for catalytic activity of OSR1, although not part of the consensus core of protein kin...

Molecular components of signal amplification in olfactory sensory cilia

“…Through their effects on SPAK/OSR1, WNK kinases control members of the SLC12 family of Cl − /cation cotransporters (NKCCs, KCCs, NCC) and thereby play a pivotal role in cellular Cl − homeostasis (39). Recent studies have revealed that WNK1 and WNK4 phosphorylate OSR1 and SPAK (40)(41)(42)(43) and thereby promote phosphorylation and activation of NKCC1. Our data show that all four kinases, SPAK, OSR1, WNK1, and WNK4, are expressed in ORN cilia, and SPAK and OSR1 are both necessary for maximal activation of the transporter.…”

WNKs: protein kinases with a unique kinase domain

“…Hypertonicity stimulates autophosphorylation of WNK1 on serine-382, activating WNK1 to phosphorylate SPAK and OSR1 (Anselmo et al, 2006;Zagorska et al, 2007). SPAK and OSR1 kinase activate NKCC1 and NKCC2 by causing phosphorylation on the transporters (Moriguchi et al, 2005;Vitari et al, 2005Vitari et al, , 2006Anselmo et al, 2006). Hyperotonicity also induces a rapid redistribution of WNK1 from cytosol to vesicular structures (Zagorska et al, 2007).…”

“…In addition to the regulation of NCC as described above, WNK1, 3 and 4 increase ion transport mediated by NKCC1 and NKCC2 (Rinehart et al, 2005;Vitari et al, 2005;Anselmo et al, 2006;Gagnon et al, 2006). WNK3 and 4 also inhibit KCCs (Vitari et al, 2005;Gagnon et al, 2006;Garzon-Muvdi et al, 2007).…”

“…Phosphorylation of NKCC1 is correlated with increased transporter activity. OSR1 (and SPAK) forms a tight complex with WNK1 and is directly activated by WNK1 through a catalytic mechanism (Moriguchi et al, 2005;Vitari et al, 2005Vitari et al, , 2006Anselmo et al, 2006). WNK4 also binds and regulates OSR1 and SPAK (Gagnon et al, 2006).…”

“…One is hypertonicity (Xu et al, 2000;Lenertz et al, 2005). Hypertonicity stimulates autophosphorylation of WNK1 on serine-382, activating WNK1 to phosphorylate SPAK and OSR1 (Anselmo et al, 2006;Zagorska et al, 2007). SPAK and OSR1 kinase activate NKCC1 and NKCC2 by causing phosphorylation on the transporters (Moriguchi et al, 2005;Vitari et al, 2005Vitari et al, , 2006Anselmo et al, 2006).…”