Activation of the Avian Erythrocyte Na-K-Cl Cotransport Protein by Cell Shrinkage, cAMP, Fluoride, and Calyculin-A Involves Phosphorylation at Common Sites

Na-K-Cl cotransport activity in duck erythrocytes increases ∼10-fold in response to osmotic cell shrinkage, norepinephrine, fluoride, or calyculin-A (an inhibitor of type-1 and -2a phosphatases). To assess whether all four stimuli promote phosphorylation of the cotransport protein and whether this phosphorylation is catalyzed by the same kinase, the cotransporter was isolated from erythrocytes by immunoprecipitation and its pattern of phosphorylation was evaluated. Each stimulus evoked proportionate increases in cotransporter activity and phosphorylation. No two stimuli in combination evoked greater activation and phosphorylation than did the more potent of the two stimuli acting alone. Phosphoamino acid analysis of the cotransport protein indicated that phosphorylation occurs at serine and threonine residues. Phosphopeptide mapping revealed a distinctive pattern of 8 major tryptic phosphopeptides, none of which were significantly phosphorylated in the unstimulated state. Maps of cotransporters activated by the four different stimuli were indistinguishable. Measurements of phosphorylation stoichiometry indicated that each cotransporter acquires ∼5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. Staurosporine, a kinase inhibitor with broad selectivity, inhibited each stimulus equipotently (IC50 ∼ 0.7 μm). Staurosporine promptly reversed cotransporter activity and phosphorylation when added to shrinkage-stimulated but not to calyculin-stimulated cells, indicating that it enters the cell rapidly and blocks phosphorylation. These results suggest that cell shrinkage, cAMP, fluoride, and calyculin-A promote the phosphorylation of the Na-K-Cl cotransport protein at a similar constellation of serine and threonine residues. It is proposed that all modes of stimulation ultimately involve the same protein kinase. Na-K-Cl cotransport activity in duck erythrocytes increases ∼10-fold in response to osmotic cell shrinkage, norepinephrine, fluoride, or calyculin-A (an inhibitor of type-1 and -2a phosphatases). To assess whether all four stimuli promote phosphorylation of the cotransport protein and whether this phosphorylation is catalyzed by the same kinase, the cotransporter was isolated from erythrocytes by immunoprecipitation and its pattern of phosphorylation was evaluated. Each stimulus evoked proportionate increases in cotransporter activity and phosphorylation. No two stimuli in combination evoked greater activation and phosphorylation than did the more potent of the two stimuli acting alone. Phosphoamino acid analysis of the cotransport protein indicated that phosphorylation occurs at serine and threonine residues. Phosphopeptide mapping revealed a distinctive pattern of 8 major tryptic phosphopeptides, none of which were significantly phosphorylated in the unstimulated state. Maps of cotransporters activated by the four different stimuli were indistinguishable. Measurements of phosphorylation stoichiometry indicated that each cotransporter acquires ∼5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. Staurosporine, a kinase inhibitor with broad selectivity, inhibited each stimulus equipotently (IC50 ∼ 0.7 μm). Staurosporine promptly reversed cotransporter activity and phosphorylation when added to shrinkage-stimulated but not to calyculin-stimulated cells, indicating that it enters the cell rapidly and blocks phosphorylation. These results suggest that cell shrinkage, cAMP, fluoride, and calyculin-A promote the phosphorylation of the Na-K-Cl cotransport protein at a similar constellation of serine and threonine residues. It is proposed that all modes of stimulation ultimately involve the same protein kinase. Na-K-Cl cotransport is regulated by numerous first and second messengers through a complex and cell-specific interplay of stimulatory and inhibitory signals (1Palfrey H.C. O'Donnell M.E. Cell Physiol. Biochem. 1992; 2: 293-307Crossref Scopus (45) Google Scholar). The molecular mechanisms by which cell surface receptors, cell volume, cytosolic chloride, cytoskeletal architecture, and proliferative status modulate cotransport activity remain unknown. Early recognition that ion movement by the Na-K-Cl cotransporter, although energetically passive (2Geck P. Pietrzyk C. Burckhardt B.-C. Pfeiffer B. Heinz E. Biochim. Biophys. Acta. 1980; 600: 432-447Crossref PubMed Scopus (339) Google Scholar, 3Haas M. Schmidt III, W.F. McManus T.J. J. Gen. Physiol. 1982; 80: 125-147Crossref PubMed Scopus (133) Google Scholar), requires cytosolic ATP and Mg2+ (2Geck P. Pietrzyk C. Burckhardt B.-C. Pfeiffer B. Heinz E. Biochim. Biophys. 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Circumstantial support came from demonstrations that cotransport activity is increased by agents that inhibit protein phosphatases (10Altamirano A.A. Breitwieser G.E. Russell J.M. Am. J. Physiol. 1988; 254: C582-C586Crossref PubMed Google Scholar, 11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar) and decreased by agents that inhibit protein kinases (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar, 12O'Grady S.M. DeJonge H.R. Vaandrager A.B. Field M. Am. J. Physiol. 1988; 254: C115-C121Crossref PubMed Google Scholar). Recent studies have established that the Na-K-Cl cotransporter itself is a phosphoprotein (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar, 13Lytle C. Forbush III, B. J. Biol. 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Physiol. 1993; PubMed Scopus Google Scholar) might cotransport Duck erythrocytes have a of ion transport by of and of These cells Na-K-Cl cotransport in response to four of osmotic cell shrinkage, cytosolic and H.C. Greengard P. Ann. N. Y. Acad. Sci. PubMed Scopus Google Scholar, H.C. and of Cell Scholar). modes of the response is with increases in H.C. Greengard P. Ann. N. Y. Acad. Sci. PubMed Scopus Google Scholar) and protein kinase activity (8Alper S.L. Beam K.G. Greengard P. J. Biol. Chem. 1980; 255: 4864-4871Abstract Full Text PDF PubMed Google Scholar, 11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar), and by kinase and at that protein kinase in erythrocytes (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar). The same kinase activation of cotransport by cell shrinkage, fluoride, and but at an of (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar). These suggest that the Na-K-Cl cotransporter is regulated by at two of which is protein kinase. The that blocks activation by cell shrinkage, fluoride, and acid at a similar the that stimuli by the same kinase H.C. and of Cell Scholar). While cotransport activity to by a protein kinase and a is whether all modes of stimulation involve phosphorylation of the cotransport protein itself and whether different stimuli involve different The of the was to the that four different modes of stimulation shrinkage, cAMP, fluoride, and involve phosphorylation of the cotransport protein at The recent of of the Na-K-Cl cotransport protein from of duck erythrocytes with C. D. Forbush III, B. Am. J. Physiol. 1995; 269: PubMed Google Scholar) it to the phosphorylation by different stimuli and to assess the of phosphorylation The results of this analysis suggest that all of activation promote phosphorylation of the cotransport protein at a of was from were from was from were from and were from were from protein kinase. membrane was from and were from the of the Na-K-Cl cotransporter was C. D. Forbush III, B. Am. J. Physiol. 1995; 269: PubMed Google Scholar). The of duck cotransporter on C. D. Forbush III, B. Am. J. 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PubMed Scopus Google Scholar). were to cotransporter activation and phosphorylation. were in and an of cotransport norepinephrine, fluoride, or for a which the of each and was by the of and by with was by the cells in and was by were to an which was to The of to cotransport was by of the to this of the and was by of cells were in The cells were in in and in of by to for the was with of and on the was by for at and on with of were on and four with by with The was of and and on Biochem. PubMed Scopus Google Scholar). was or a analysis indicated that the of immunoprecipitation from of both and cells of ATP in acid of cells was by on in at M. B. J. PubMed Google Scholar). To of with were from the and by for a of for to (8Alper S.L. Beam K.G. Greengard P. J. Biol. Chem. 1980; 255: 4864-4871Abstract Full Text PDF PubMed Google Scholar). The of in was by and J. Biol. Chem. Full Text PDF PubMed Google Scholar). acid of cells was to a of The was with of and ATP was with in The was in of and The was in of of to ATP was to by and of and at for The was to a and the was with of was with which was with in were for by of for the of ATP and for the of and of The of to the of by was to The activity of was from the of ATP in the the the of cells the of the duck C. and K-Cl Cotransport in Duck Red Cells. Ph.D. thesis, Duke University, Durham, NC.Google Scholar), and the of ATP the duck 7Palfrey H.C. Pewitt E.B. Pflügers Arch. 1993; 425: 321-328Crossref PubMed Scopus (38) Google Scholar). protein was isolated from cells by by and to of were by and in acid at for The was in of of and by for at on a in a of and Phosphoamino were by and a were with with for at to D. 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Ph.D. thesis, Duke University, Durham, NC.Google of ATP in duck were with at for the indicated The activity of in ATP was from to cotransport activity norepinephrine, fluoride, and of the cotransport protein phosphorylation was with that cotransport fluoride, and H.C. and of Cell Scholar). added with the cotransporter activity but did not the or the of phosphorylation not this the that cotransporter phosphorylation is the than the of cotransport activity and the that by phosphorylation. The stimulatory of was not by the of by the of with not the that it is to proposed for cells M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The of changes in cell on cotransporter activity and phosphorylation was on of duck The for at on cell or on the to unstimulated cells, cotransporter activity of osmotic cell from to of cell a of cotransport activity and a in cotransport protein phosphorylation of to in cotransporter phosphorylation. phosphorylation when the swollen erythrocytes were for to a broad protein kinase inhibitor that rapidly cotransport activity results suggest that the cotransport protein a of or inhibitory phosphorylation whose is and or of Na-K-Cl cotransport protein phosphorylation a of cell were with for to an or from each cell were for analysis of cell cell and protein of each of cotransport protein cell cell results from four of four stimuli on Na-K-Cl cotransporter activity and of of duck erythrocytes with were for to or in at or calyculin-A Cotransport activity was and was of cotransport protein at were to with calyculin-A Each the in four to of activation and phosphorylation of Na-K-Cl cotransporter cell and by in duck erythrocytes was at and the of the a cotransport protein phosphorylation was the same the was by the of to was added to of Na-K-Cl cotransporter activation and kinase duck erythrocytes were for at in a or the was with or was each in for and the of the with activation by cell or by with duck erythrocytes were for at with calyculin-A or was added to was each in for and the of the reversed activation evoked by cell but not that evoked by equipotently blocks activation of Na-K-Cl cotransport by norepinephrine, cell shrinkage, and Duck erythrocytes were in a at the an of cotransport norepinephrine, fluoride, or was and the of was the The of was to that evoked by the stimulus in the of inhibitor Each the of in which were in The of each stimulus was inhibited by Cell evoked increases in cotransporter activity and phosphorylation in cell from to of cell evoked transport The of cell on phosphorylation was for the cotransporter none of the major membrane of which on a were significantly by osmotic not the phosphorylation is the of transport each cotransport activity at To the stoichiometry of of erythrocytes were for cell activity, and cotransport protein phosphorylation the of the cotransport protein by immunoprecipitation and The of cotransporters in the was from the of on a duck the of erythrocytes to immunoprecipitation and the of immunoprecipitation The of with the cotransporter was from the of the cotransporter and the activity of the The results of four in which the cell and phosphorylation stoichiometry was is in cells of volume, the active cotransporter this to increased it to The analysis that each cotransporter acquires phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. analysis that each cotransporter a of activation M. Forbush III, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and that to to a The of is not the for a of cotransport to a of a stimulus III, B. Forbush J. Biol. 1994; Scholar). the cotransporters to similar of cotransport evoked stimuli in or in The analysis that all cotransporters and is this is by the that duck cotransporters on and of cotransporter activity and phosphorylation indicated that norepinephrine, fluoride, and when at evoked similar of cotransport activity and cotransport protein phosphorylation norepinephrine, activity increased and this was with a in phosphorylation. The response was by a of not with that by (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar, H.C. Greengard P. Ann. N. Y. Acad. Sci. PubMed Scopus Google Scholar). Coordinate increases in activity and phosphorylation were in response to stimuli that not involve shrinkage, fluoride, and activity and phosphorylation with an at and at not The of activity and phosphorylation were evoked by the of the four stimuli were not of two stimuli in combination evoked more activity or phosphorylation than did the more potent of the two stimuli acting alone. suggests that the four stimuli the cotransport protein by its phosphorylation at regulatory Cell increased cotransporter activity and phosphorylation to of activity and phosphorylation on of a protein kinase inhibitor with broad Biochem. 1994; Google Scholar). of the activation and was by the cells to activation in response to cell or calyculin-A a and to a by calyculin-A was ∼ than that by cell ∼ to cells, the cotransporter to cell and calyculin-A and to and not added to cells by osmotic shrinkage, cotransporter activity and phosphorylation to rapidly was not in cells with calyculin-A indicating that the cotransporter by its phosphorylation than by its each of the four stimuli to by a kinase that is or by all modes of activation involve the same each stimulus inhibited by To this erythrocytes were for with of stimulation by norepinephrine, fluoride, or in all stimuli were inhibited to a by a similar of μm). To assess the of phosphorylation the cotransport protein was with an that D. Biochem. 1982; PubMed Scopus Google Scholar). of cells with the cotransporter was isolated by immunoprecipitation and the cotransporter were with were by and for two major of and The to different of were with and with for an additional to the isolated the and a that the of the cotransport protein C. D. Forbush III, B. Am. J. Physiol. 1995; 269: PubMed Google Scholar) the on of protein not all four each of the two was phosphorylated to similar from calyculin-stimulated cotransporters greater phosphorylation and and which changes in the or with the of phosphorylation. These results each stimulus phosphorylation of two of which of the Phosphoamino acid analysis of the cotransport protein indicated that each of the four stimuli promote phosphorylation at serine and threonine residues. was not in or of cotransporters isolated from erythrocytes revealed a distinctive pattern of tryptic in and Maps of cotransporters phosphorylated in response to cell shrinkage, fluoride, and were of the were in of unstimulated cotransporters calyculin-A evoked more cotransporter phosphorylation and activity than did stimuli and it was to whether the inhibitor phosphorylation of different or more phosphorylation of the same To of cotransporters by cell calyculin-A were The of with cell and calyculin-A were similar and with and No additional were in of cotransporters by cell and calyculin-A the greater of cotransporter phosphorylation and activity with calyculin-A to a more phosphorylation of the same phosphorylated with each a phosphorylation is might of or of different or a with phosphorylation is cotransporters were with in and different the of each major of in two is It is that activation a of tryptic phosphorylated and that a similar is with each The suggest that of Na-K-Cl cotransport in the shrinkage, cAMP, fluoride, and promote phosphorylation of the cotransport protein at a constellation of serine and threonine residues. These results the that the cotransport protein is regulated by phosphorylation (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar, 13Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 14Torchia J. Lytle C. Pon D.J. Forbush III, B. Sen A.K. J. Biol. Chem. 1992; 267: 25444-25450Abstract Full Text PDF PubMed Google Scholar) and suggest that different stimuli through the same kinase. The kinase is by of of two stimuli in combination greater cotransport activity or phosphorylation than the more potent stimulus the phosphorylation evoked the four stimuli was catalyzed by different kinases acting on of the stimuli in the of the phosphorylation by the stimuli The that phosphorylation by stimulus phosphorylation by a different and suggest that the four stimuli on of cotransporters activated by the four different stimuli and equipotently blocks activation of the cotransporter by cell shrinkage, cAMP, and with the that each signal is to the same by the same kinase. The molecular on the cotransport protein that ion to involve serine and threonine residues. of phosphorylation stoichiometry that the cotransport protein acquires ∼5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells, and a of tryptic studies that the is and of the cotransport the cotransporter two and whose that of the protein all four of the different the molecular of the cotransporter is of the Na-K-Cl cotransporter all of which Forbush III, B. Cell Biol. 1995; PubMed Scopus Google Scholar). the cotransporter the same the It is that the and phosphorylated by at residues. both each that in The of major phosphorylation in and on the cells of the cells, in duck the Na-K-Cl cotransporter is and phosphorylated by cell shrinkage, cAMP, and calyculin-A at threonine and serine (13Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 18Lytle C. Forbush III, B. Am. J. Physiol. 1996; 270: C437-C448Crossref PubMed Google Scholar). of the threonine have in the that to cell and in the that to (13Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar). of the cotransporter, including that to the for the that the of is that all stimuli the cotransporter the same and suggests that the cotransporter in two and a a kinase and a The at which state to state of calyculin-A that the kinase and active and that the the kinase in unstimulated cells H.C. Pewitt E.B. Pflügers Arch. 1993; 425: 321-328Crossref PubMed Scopus (38) Google Scholar, 11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar). this is more to by calyculin-A than to acid H.C. Pewitt E.B. Pflügers Arch. 1993; 425: 321-328Crossref PubMed Scopus (38) Google Scholar), it to type-1 is to inhibited by protein kinase in and by in P. Biochem. PubMed Scopus Google Scholar), it is that and fluoride, the cotransporter by its cells changes in and transport that is is that the signal cotransport protein but whether the signal the kinase, the or both and N. J. Gen. Physiol. 1990; PubMed Scopus Google Scholar) from the of K-Cl cotransport in erythrocytes that cell phosphorylation than and the shrinkage-stimulated kinase with studies on duck which that with Na-K-Cl cotransporters active and phosphorylated more the cells activation of in is by cell L. M. C. J. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the than its stimulation by cell and to kinase suggests that the signal might modulate Palfrey and Pewitt H.C. Pewitt E.B. Pflügers Arch. 1993; 425: 321-328Crossref PubMed Scopus (38) Google Scholar) that cotransporters activated by cell shrinkage, activated by cAMP, to kinase of or by of ATP or and that cell might this was not by the with this kinase inhibitor was added to shrunken cells, cotransport activity and cotransport protein phosphorylation rapidly that the active cell the of the signal not to protein kinase protein kinase activity, or protein kinase of kinases and cytosolic have on the phosphorylation activity, or of the cotransport protein in duck The recent of kinase a shrinkage-stimulated kinase (16Klein J.D. O'Neill W.C. Am. J. Physiol. 1995; 269: C1524-C1531Crossref PubMed Google Scholar, M. N. Am. J. Physiol. 1993; Google Scholar) prompted and O'Neill to suggest that the signal to the cotransport was on two changes in cells evoked in Na-K-Cl cotransport activity and and both were inhibited equipotently by the inhibitor (16Klein J.D. O'Neill W.C. Am. J. Physiol. 1995; 269: C1524-C1531Crossref PubMed Google Scholar). did not phosphorylation of the cotransport protein in response to cell shrinkage, that the of on the cotransporter is through in cytoskeletal (16Klein J.D. O'Neill W.C. Am. J. Physiol. 1995; 269: C1524-C1531Crossref PubMed Google Scholar). that signal not is that duck erythrocytes of in to cell is for stimulation of cotransport by cell and occurs a in cytosolic (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar, H.C. Greengard P. Ann. N. Y. Acad. Sci. PubMed Scopus Google Scholar), stimulation by with acid is not with increased activity (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar), stimulation by cell in the of the kinase inhibitor at that activity (11Pewitt E.B. Hegde R.S. Haas M. Palfrey H.C. J. Biol. Chem. 1990; 265: 20747-20756Abstract Full Text PDF PubMed Google Scholar), and the Na-K-Cl cotransporter of the activated and phosphorylated in response to (13Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar) although it a for Lytle C. E. Forbush III, B. Acad. Sci. A. 1994; PubMed Scopus Google Scholar). calyculin-A greater cotransporter phosphorylation and activity than the this to a more phosphorylation of the same that phosphorylated with the is on the that additional phosphorylation occurs when calyculin-stimulated cells to and that of cotransporters phosphorylated by the stimuli it that each phosphorylated is by a support of this calyculin-A in in It is not of the kinase, by the cotransporters the phosphorylated state. is whether phosphorylation of the cotransport protein is both necessary and sufficient for The that blocks cotransport protein phosphorylation the that phosphorylation is in modes of regulation that not involve phosphorylation a recent of the revealed that of cotransport activity and increases in cotransport protein shrinkage, and not A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). modes of regulation not in cells (13Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 18Lytle C. Forbush III, B. Am. J. Physiol. 1996; 270: C437-C448Crossref PubMed Google Scholar), or duck erythrocytes osmotic and calyculin-A promote cotransport protein phosphorylation. results suggest that the cotransport protein is phosphorylated at a of serine and threonine in response to cell shrinkage, cAMP, fluoride, and by results the for kinases acting on of the cotransport protein and for a more in which cotransport activity on the of kinase and and for and A. and for