p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1.

The Na+/H+ exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90(RSK)) is a key NHE-1 kinase since p90(RSK) phosphorylates NHE-1 serine 703 stoichiometrically in vitro, and transfection with kinase-inactive p90(RSK) inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90(RSK) as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

[1]  J. Avruch,et al.  Regulation of an epitope-tagged recombinant Rsk-1 S6 kinase by phorbol ester and erk/MAP kinase. , 1993, Biochemistry.

[2]  J. Feramisco,et al.  Isolation of phosphorylated peptides and proteins on ion exchange papers. , 1978, Analytical biochemistry.

[3]  E. Van Obberghen,et al.  Co-regulation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor. , 1993, The Journal of biological chemistry.

[4]  L. Ng,et al.  Sodium–hydrogen antiporter protein in normotensive Wistar–Kyoto rats and spontaneously hypertensive rats , 1994, Journal of hypertension.

[5]  S. Shenolikar,et al.  Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na(+)-H+ exchanger. , 1995, The Journal of clinical investigation.

[6]  J. Pouysségur,et al.  Growth factor activation and "H(+)-sensing" of the Na+/H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation. , 1994, The Journal of biological chemistry.

[7]  J. Maller,et al.  Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II , 1988, Nature.

[8]  J. Pouysségur,et al.  Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective. , 1994, The Journal of biological chemistry.

[9]  S. Grinstein,et al.  Na+/H+ Exchangers of Mammalian Cells* , 1997, The Journal of Biological Chemistry.

[10]  S. Grinstein,et al.  Muscarinic Agonists Induce Phosphorylation-independent Activation of the NHE-1 Isoform of the Na+/H+ Antiporter in Salivary Acinar Cells* , 1997, The Journal of Biological Chemistry.

[11]  R. Alexander,et al.  Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange. , 1989, The Journal of clinical investigation.

[12]  L. Fliegel,et al.  Phosphorylation of the C-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II. , 1992, The Biochemical journal.

[13]  S. Grinstein,et al.  Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism. , 1992, The Journal of biological chemistry.

[14]  C. Sardet,et al.  Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites. , 1991, The Journal of biological chemistry.

[15]  Y. Granot,et al.  Stimulation of Mitogen-activated Protein Kinase and Na+/H+ Exchanger in Human Platelets , 1996, The Journal of Biological Chemistry.

[16]  R. Tsien,et al.  Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts , 1983, Nature.

[17]  D. Barber,et al.  A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. Cohen,et al.  Identification of Regulatory Phosphorylation Sites in Mitogen-activated Protein Kinase (MAPK)-activated Protein Kinase-1a/p90 rsk That Are Inducible by MAPK* , 1998, The Journal of Biological Chemistry.

[19]  J. Abe,et al.  Angiotensin II stimulates p90rsk in vascular smooth muscle cells. A potential Na(+)-H+ exchanger kinase. , 1997, Circulation research.

[20]  E. Chang,et al.  Expression and phosphorylation of NHE1 in wild‐type and transformed human and rodent fibroblasts , 1994, Journal of cellular physiology.

[21]  A. Bridges,et al.  A synthetic inhibitor of the mitogen-activated protein kinase cascade. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  B. Berk,et al.  A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells. , 1997, Hypertension.

[23]  J. Pouysségur,et al.  Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0----G1 transition of fibroblasts. , 1986, Experimental cell research.

[24]  B. Berk,et al.  Long-term regulation of Na(+)-H+ exchange in vascular smooth muscle cells: role of protein kinase C. , 1991, The American journal of physiology.

[25]  J. Pouysségur,et al.  Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+. , 1984, The Journal of biological chemistry.

[26]  J. Maller,et al.  Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6. , 1986, The Journal of biological chemistry.

[27]  Jiahuai Han,et al.  p38 Kinase is a negative regulator of angiotensin II signal transduction in vascular smooth muscle cells: effects on Na+/H+ exchange and ERK1/2. , 1998, Circulation research.

[28]  L. Ng,et al.  Activity and expression of Na(+)-H+ exchanger isoforms 1 and 3 in kidney proximal tubules of hypertensive rats. , 1997, Circulation research.

[29]  W. Siffert,et al.  Sodium-proton exchange and primary hypertension. An update. , 1995, Hypertension.

[30]  L. Ng,et al.  Na + -H + Exchanger Isoform 1 Phosphorylation in Normal Wistar-Kyoto andSpontaneously Hypertensive Rats , 1995 .

[31]  J. Pouysségur,et al.  The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites. , 1994, The Journal of biological chemistry.

[32]  S. Grinstein,et al.  ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization. , 1994, The Journal of biological chemistry.

[33]  R. Aebersold,et al.  Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. , 1994, The Journal of biological chemistry.

[34]  C. Sardet,et al.  The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H(+)-sensing". , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Alexander,et al.  Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways. , 1987, The Journal of biological chemistry.

[36]  S. Grinstein,et al.  Coimmunoprecipitation of a 24-kDa protein with NHE1, the ubiquitous isoform of the Na+/H+ exchanger. , 1996, The American journal of physiology.

[37]  C. Sardet,et al.  A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[38]  D. Barber,et al.  p160ROCK mediates RhoA activation of Na–H exchange , 1998, The EMBO journal.

[39]  B. Berk,et al.  Na(+)-H+ exchanger expression in vascular smooth muscle of spontaneously hypertensive and Wistar-Kyoto rats. , 1994, Hypertension.

[40]  S. Gygi,et al.  Electrophoresis combined with novel mass spectrometry techniques: Powerful tools for the analysis of proteins and proteomes , 1998, Electrophoresis.

[41]  S. Shenolikar,et al.  The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange , 1998, Nature.

[42]  J. Pouysségur,et al.  The p42/p44 Mitogen-activated Protein Kinase Cascade Is Determinant in Mediating Activation of the Na+/H+ Exchanger (NHE1 Isoform) in Response to Growth Factors* , 1997, The Journal of Biological Chemistry.

[43]  S. Pelech,et al.  Phosphorylation and regulation of the Na+/H+ exchanger through mitogen-activated protein kinase. , 1997, Biochemistry.

[44]  C. Sardet,et al.  Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. , 1990, Science.

[45]  W. Frankel,et al.  Sodium/Hydrogen Exchanger Gene Defect in Slow-Wave Epilepsy Mutant Mice , 1997, Cell.

[46]  G. Shull,et al.  Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins. , 1992, The Journal of biological chemistry.

[47]  C. Sardet,et al.  Functional expression of the human growth factor activatable Na+/H+ antiporter (NHE-1) in baculovirus-infected cells. , 1991, Biochemistry.

[48]  E. Krebs,et al.  Deletion of 11 Amino Acids in p90rsk-mo-1Abolishes Kinase Activity , 1999, Molecular and Cellular Biology.

[49]  J. Pouysségur,et al.  Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. , 1995, The American journal of physiology.