Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

Abstract

The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.T he protein kinases oxidative stress-responsive 1 (OSR1) and its homolog the STE20/SPS1-related proline-, alanine-rich kinase (SPAK or PASK) are the mammalian members of the germ-cell kinase VI subgroup of the large Ste20 branch of the mammalian kinome. OSR1 and SPAK directly regulate the solute carrier 12 family of cation-chloride cotransporters which modulate ion homeostasis throughout the body (1, 2). OSR1/ SPAK kinase domains lie close to their N-termini and they contain two additional conserved regions named "PF1" and "PF2" [PASK and Fray (Drosophila homolog)] (3). PF1 is a C-terminal extension to the kinase domain and is required for enzyme activity (4). PF2 binds the consensus motif [(R/K)FX(V/I)] (5) in substrates including ion cotransporters and in regulators. OSR1 and SPAK are activated by with no lysine [K] (WNK) protein kinases, which phosphorylate the essential activation loop regulatory site as well as a second site in the PF1 region with an undefined function (6-9).The four WNK protein kinases are large enzymes notable for the alternative placement of the essential ATP-binding lysine residue in their catalytic domains, distinguishing them from other members of the protein kinase superfamily (10, 11). Initial attention was focused on these enzymes because certain mutations in two family members cause pseudohypoaldosteronism type II, a heritable form of hypertension (12). WNKs are activated by changes in tonicity. Cellular reconstitution studies and mouse genetics demonstrated the importance of WNK function in cell volume regulation and maintenance of blood pressure (13-19). Control of cation-chloride cotransporters through OSR1 and SPAK is among the best-documented actions of WNKs in diverse tissues (5,(20)(21)(22).WNKs also regulate serum-and glucocorticoid-inducible protein k...

Actions of the protein kinase WNK1 on endothelial cells are differentially mediated by its substrate kinases OSR1 and SPAK

“…− cotransporters 1 and 2 (NKCC1 and NKCC2), and K + , Cl − cotransporters (KCCs) (7)(8)(9)(11)(12)(13)(14)(15)(16)(17). Although WNK1 +/− heterozygous mice are viable (18,19), a study using whole-embryo and endothelialspecific WNK1 knockout mice showed embryonic lethality due to angiogenic and cardiovascular defects (20,21).…”

Structural and Biochemical Insights into the Activation Mechanisms of Germinal Center Kinase OSR1

“…reported that osmotic stress, such as hyperosmotic stress induced by sorbitol, stimulates the activation of OSR1 (8,21). The activated OSR1 phosphorylates members of ion cotransporters to maintain cell volume homeostasis.…”

“…Mutational analysis indicates that phosphorylation of Thr-185 in OSR1 could significantly enhance its kinase activity, while phosphorylation of Ser-325 only modestly promotes OSR1 kinase activity (19). Moreover, the mammalian target of rapamycin complex 2 (mTORC2), which is activated by phosphatidylinositol-3 kinase (PI3K), can increase OSR1 activity by phosphorylating residue Ser-339 (21,22). In addition, OSR1 has also been implicated in immune regulation by interacting with protein kinase C-(PKC) or TNF receptor expressed in lymphoid tissues (RELT) homologues (18,23).…”

Downregulation of Peptide Transporters PEPT1 and PEPT2 by Oxidative Stress Responsive Kinase OSR1

“…The oxidative-stress-responsive kinase 1 (OSR1) is engaged in the regulation of epithelial transport [1,2,3,4]. OSR1 and the related SPAK (STE20-related proline/alanine-rich kinase) upregulate the NaCl (NCC) and Na + ,K + ,2Cl - (NKCC2) cotransporters and thus impact on blood pressure [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17].…”

“…OSR1 and the related SPAK (STE20-related proline/alanine-rich kinase) upregulate the NaCl (NCC) and Na + ,K + ,2Cl - (NKCC2) cotransporters and thus impact on blood pressure [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. By stimulating cellular ion uptake SPAK and OSR1 contribute to cell volume regulation [9,18].…”