Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals Samarpita Senguptaa, Andrés Lorente-Rodrígueza, Svetlana Earnesta, Steve Stippeca, Xiaofeng Guob, David C. Trudgianb, Hamid Mirzaeib, and Melanie H. Cobba,1 Departments of aPharmacology and bBiochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041 Contributed by Melanie H. Cobb, October 3, 2013 (sent for review October 6, 2012) The Ste20 family protein kinases oxidative stress-responsive 1 (ENaC) (23, 24). SGKs and the related Akt enzymes are acti- (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase vated by phosphorylation on multiple sites, most prominently a directly regulate the solute carrier 12 family of cation-chloride residue in the activation loop by the phosphoinositide-dependent cotransporters and thereby modulate a range of processes includ- protein kinase and on a second site in a C-terminal hydrophobic ing cell volume homeostasis, blood pressure, hearing, and kidney motif (25). The kinase that phosphorylates the hydrophobic function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase motif site under many circumstances is the mammalian target are activated by with no lysine [K] protein kinases that phosphory- of rapamycin complex 2 (mTORC2), which provides an addi- late the essential activation loop regulatory site on these kinases. tional phosphatidylinositol-3 kinase (PI3K)-dependent input to We found that inhibition of phosphoinositide 3-kinase (PI3K) these kinases (26–33). reduced OSR1 activation by osmotic stress. Inhibition of the PI3K In this study, we show that OSR1 is phosphorylated not only target pathway, the mammalian target of rapamycin complex 2 by WNKs but also on a C-terminal site, conserved in SPAK, by (mTORC2), by depletion of Sin1, one of its components, decreased mTORC2. These studies reveal a link between WNK-OSR1/SPAK activation of OSR1 by sorbitol and reduced activity of the OSR1 and the PI3K-mTORC2 cascade that suggests that OSR1 and substrate, the sodium, potassium, two chloride cotransporter, in SPAK integrate signals from osmosensing and survival pathways. HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, Results and mutation of this residue eliminated OSR1 phosphorylation by OSR1 Is Regulated by a PI3K-Dependent Mechanism. In response to mTORC2. Thus, we identify a previously unrecognized connection changes in tonicity, OSR1 is activated through phosphorylation of the PI3K pathway through mTORC2 to a Ste20 protein kinase and of its activation loop by WNKs. Previously, we failed to find ion homeostasis. activation of OSR1 by serum or epidermal growth factor (4). A recent report, however, indicates that it participates in insulin- phosphoregulation | Akt | ion transport | WNK1 regulated events in a PI3K-sensitive manner (34). To confirm the PI3K sensitivity and retest potential regulation by growth factors, he protein kinases oxidative stress-responsive 1 (OSR1) and we examined effects of the PI3K inhibitor wortmannin on OSR1 Tits homolog the STE20/SPS1-related proline-, alanine-rich activity in HeLa cells stimulated with sorbitol or serum. The kinase (SPAK or PASK) are the mammalian members of the sodium, potassium, two chloride cotransporters 1 and 2 (NKCC1 germ-cell kinase VI subgroup of the large Ste20 branch of the and NKCC2) are related ion cotransporters of the solute carrier mammalian kinome. OSR1 and SPAK directly regulate the sol- 12 family that are phosphorylated and activated by OSR1/SPAK ute carrier 12 family of cation-chloride cotransporters which (2, 15). The activity of immunoprecipitated OSR1 was measured modulate ion homeostasis throughout the body (1, 2). OSR1/ using a recombinant N-terminal fragment of NKCC2, residues – SPAK kinase domains lie close to their N-termini and they con- 1 175, as substrate (9). Sorbitol stimulated OSR1 activity, but A tain two additional conserved regions named “PF1” and “PF2” serum did not, consistent with earlier results (Fig. 1 ). Wortmannin [PASK and Fray (Drosophila homolog)] (3). PF1 is a C-terminal decreased serum-stimulated phosphorylation of Akt on its hydro- extension to the kinase domain and is required for enzyme ac- phobic motif site and also reduced sorbitol-stimulated OSR1 kinase tivity (4). PF2 binds the consensus motif [(R/K)FX(V/I)] (5) in substrates including ion cotransporters and in regulators. OSR1 Significance and SPAK are activated by with no lysine [K] (WNK) protein kinases, which phosphorylate the essential activation loop regu- With no lysine [K] (WNK) protein kinases are sensitive to latory site as well as a second site in the PF1 region with an un- changes in osmotic stress. Through the downstream protein defined function (6–9). kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1- The four WNK protein kinases are large enzymes notable for related proline-, alanine-rich kinase, WNKs regulate a family of the alternative placement of the essential ATP-binding lysine ion cotransporters and thereby modulate a range of processes residue in their catalytic domains, distinguishing them from other including cell volume homeostasis, blood pressure, hearing, members of the protein kinase superfamily (10, 11). Initial at- and kidney function. We found that a major phosphoinositide tention was focused on these enzymes because certain mutations 3-kinase target pathway, the mammalian target of rapamycin in two family members cause pseudohypoaldosteronism type II, a complex 2, also phosphorylates OSR1, coordinating with WNK1 heritable form of hypertension (12). WNKs are activated by to enhance OSR1 and ion cotransporter function. changes in tonicity. Cellular reconstitution studies and mouse genetics demonstrated the importance of WNK function in cell Author contributions: S. Sengupta, S.E., D.C.T., H.M., and M.H.C. designed research; – S. Sengupta, A.L.-R., S.E., S. Stippec, and X.G. performed research; X.G., D.C.T., and H.M. volume regulation and maintenance of blood pressure (13 19). contributed new reagents/analytic tools; S. Sengupta, A.L.-R., S.E., S. Stippec, D.C.T., and H.M. Control of cation-chloride cotransporters through OSR1 and analyzed data; and S. Sengupta, A.L.-R., and M.H.C. wrote the paper. SPAK is among the best-documented actions of WNKs in diverse The authors declare no conflict of interest. – tissues (5, 20 22). 1To whom correspondence should be addressed. E-mail: melanie.cobb@utsouthwestern. WNKs also regulate serum- and glucocorticoid-inducible pro- edu. tein kinases (SGKs) through a noncatalytic mechanism leading This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to increased sodium influx through the epithelial sodium channel 1073/pnas.1318676110/-/DCSupplemental. 18826–18831 | PNAS | November 19, 2013 | vol. 110 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1318676110 Downloaded by guest on September 28, 2021 A OSR1 Is Regulated by WNK1 and mTORC2. Because of the input of 5 *** mTORC2 to SGK (27), we tested the possibility that mTORC2 4 might be responsible for the PI3K-dependence of OSR1 acti- 3 vation by sorbitol. To evaluate the contribution of mTORC2, we inhibited its activity by depleting Sin1, a required component of 2 complex 2 (26). Knockdown of Sin1 reduced activation of OSR1 relative units 1 by sorbitol (Fig. 1B), supporting the conclusion that mTORC2 is OSR1 kinase activity, 0 a PI3K-dependent input to OSR1. In contrast to NKCC2 which Control Sorbitol FBS Wortmannin - + - + - + displays tissue-restricted expression in kidney, NKCC1 is expressed Kinase in most cell types including HeLa. To test the consequences of assay NKCC Sin1 depletion on cation-chloride cotransporter function, we OSR pAkt * A 120 B 5 ** 100 OSR 4 NKCC 80 3 60 relative units 2 40 OSR1 kinase activity, 1 Fold kinase activity 20 0 Sorbitol - - + + - - + + 0 siControl siSin1 Sorbitol - + + + Rapa KU Kinase assay Sin1 BIOCHEMISTRY OSR1 ERK1/2 NKCC Akt pAkt B C ** Rb uptake 86 WNK1 kinase activity, % WNK1 Fold bumetanide-sensitive Fold bumetanide-sensitive Kinase assay Fig. 1. PI3K pathways influence OSR1 activation and function in response to sorbitol. (A) HeLa cells were pretreated with 50 nM wortmannin (Wort) and GST-OSR1KR then stimulated with 0.5 M sorbitol or 20% FBS to stimulate Akt. Endogenous WNK1 OSR1 was immunoprecipitated and its activity measured using recombinant Sin1- NKCC2 1–175. Four such experiments were quantified (Top). A representative autoradiogram is shown with immunoblots of OSR1 and Akt phosphorylated on S473. Phosphorylation of Akt at S473 measured as an indicator of mTORC2 Fig. 2. Inhibition of mTORC2 reduces OSR1 activity. (A) HeLa cells were activity and pAkt as a positive control of stimulation conditions. n = 4, one pretreated for 15 min with either 100 nM rapamycin (Rapa) or 300 nM KU- way ANOVA, P < 0.0001; ***P = 0.0003 (adjusted from Tukey’s test). Error 0063794 (KU) and then stimulated with 0.5 M sorbitol (Sorb). Endogenous bars show standard deviation. (B) Effect of depletion of endogenous mTORC2 OSR1 was immunoprecipitated and its autophosphorylation and phosphor- by knockdown of Sin1 on OSR1 autophosphorylation (white bars) and activity ylation of recombinant NKCC2 1–175 were measured by immune-complex toward substrate (gray bars) upon sorbitol stimulation. n = 5, **P < 0.05. Error kinase assay. The incorporated radioactivity was measured by scintillation bars show standard error. Immunoblots show efficiency of Sin1 knockdown. counting and normalized to the amount of OSR1 immunoprecipitated in ERK1/2 was used as the loading control. Expression of Sin1 was normalized to each case. Cells stimulated with sorbitol without any drugs and without ERK1/2 expression (Bottom). (C) HeLa cells were transfected with siRNA for any other treatment were used as controls. Four experiments with 10 rep- Sin1 and scrambled siRNA as control (siC). Bumetanide sensitive uptake of licates were used to calculate standard error (error bars). *P < 0.05. Immu- 86Rb was measured as an assay of NKCC1 activity.