A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

Abstract

Phospholamban (PLB) can be phosphorylated at Ser16 by cyclic AMP-dependent protein kinase and at Thr17 by Ca2+-calmodulin-dependent protein kinase during β-agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr17 phosphorylation and attenuation of the β-agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Cardiac myocyte basal mechanics and Ca2+ kinetics were similar among the three groups. Isoproterenol stimulation was associated with phosphorylation of both Ser16 and Thr17 in wild-type PLB and Ser16 phosphorylation in T17A mutant PLB, whereas there was no detectable phosphorylation of S16A mutant PLB. Phosphorylation of Ser16 alone in T17A mutant PLB resulted in responses of the mechanical and Ca2+ kinetic parameters to isoproterenol similar to those in wild-type myocytes, which exhibited dual-site PLB phosphorylation. However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser16 in PLB can be phosphorylated independently of Thr17 in vivo, and phosphorylation of Ser16 is sufficient for mediating the maximal cardiac responses to β-adrenergic stimulation. Phospholamban (PLB) can be phosphorylated at Ser16 by cyclic AMP-dependent protein kinase and at Thr17 by Ca2+-calmodulin-dependent protein kinase during β-agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr17 phosphorylation and attenuation of the β-agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Cardiac myocyte basal mechanics and Ca2+ kinetics were similar among the three groups. Isoproterenol stimulation was associated with phosphorylation of both Ser16 and Thr17 in wild-type PLB and Ser16 phosphorylation in T17A mutant PLB, whereas there was no detectable phosphorylation of S16A mutant PLB. Phosphorylation of Ser16 alone in T17A mutant PLB resulted in responses of the mechanical and Ca2+ kinetic parameters to isoproterenol similar to those in wild-type myocytes, which exhibited dual-site PLB phosphorylation. However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser16 in PLB can be phosphorylated independently of Thr17 in vivo, and phosphorylation of Ser16 is sufficient for mediating the maximal cardiac responses to β-adrenergic stimulation. phospholamban cyclic AMP sarcoplasmic reticulum cAMP-dependent protein kinase Ca2+-calmodulin-dependent protein kinase polymerase chain reaction kilobase pair(s) α-myosin heavy chain polyacrylamide gel electrophoresis Phospholamban (PLB)1 is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLB is an inhibitor of the affinity of SERCA2 for Ca2+, and phosphorylation of PLB during β-adrenergic stimulation relieves its inhibitory effects on SERCA2 (1Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (294) Google Scholar, 2Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar). The physiological importance of PLB has been elucidated through the generation of genetically engineered mouse models with alterations in cardiac PLB expression levels (3Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Crossref PubMed Scopus (632) Google Scholar, 4Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W., II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Crossref PubMed Scopus (276) Google Scholar). Ablation of PLB was associated with significantly enhanced Ca2+ affinity of SERCA2 and myocardial performance (3Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Crossref PubMed Scopus (632) Google Scholar, 5Luo W. Wolska B.M. Grupp I.L. Harrer J.M. Haghighi K. Ferguson D.G. Slack J.P. Grupp G. Doetschman T. Solaro R.J. Kranias E.G. Circ. Res. 1996; 78: 839-847Crossref PubMed Scopus (104) Google Scholar, 6Hoit B.D. Khoury S.F. Kranias E.G. Ball N. Walsh R.A. Circ. Res. 1995; 77: 632-637Crossref PubMed Scopus (243) Google Scholar). The elevated basal contractile parameters could be minimally stimulated by β-agonists (3Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Crossref PubMed Scopus (632) Google Scholar, 7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), whereas there were no alterations in the β-receptor signaling pathway or the phosphorylation states of other major cardiac phosphoproteins (8Kiss E. Edes I. Sato Y. Luo W. Liggett S.B. Kranias E.G. Am. J. Physiol. 1997; 272: H785-H790PubMed Google Scholar). On the other hand, overexpression of PLB was associated with significant depression of contractile parameters, which could be reversed upon phosphorylation of PLB during β-agonist stimulation (4Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W., II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Crossref PubMed Scopus (276) Google Scholar). These results indicate that PLB is a key regulator of cardiac function and a prominent mediator of the β-adrenergic effects in the myocardium. In vitro studies have shown that PLB can be phosphorylated on Ser10 by protein kinase C, Ser16 by cAMP-dependent protein kinase (PKA), and Thr17by Ca2+-calmodulin-dependent protein kinase (CaMKII) (1Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (294) Google Scholar, 9Movsesian M.A. Nishikawa M. Adelstein R.S. J. Biol. Chem. 1984; 259: 8029-8032Abstract Full Text PDF PubMed Google Scholar, 10Simmerman H.K. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 261: 13333-13341Abstract Full Text PDF PubMed Google Scholar). Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca2+.In vivo studies have shown that only Ser16 and Thr17 are phosphorylated in cardiac myocytes or perfused hearts (11Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, 12Talosi L. Edes I. Kranias E.G. Am. J. Physiol. 1993; 264: H791-H797PubMed Google Scholar), whereas phosphorylation of PLB by protein kinase C has not been detected in vivo. Phosphorylation of PLB by PKA and CaMKII occurs during β-agonist exposure, although the relative contribution of each phosphorylation to the cardiac stimulatory effects is not presently clear. Each phosphorylation appears to occur independently of the other (13Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (91) Google Scholar, 14Tada M. Inui M. Yamada M. Kadoma M. Kuzuya T. Abe H. Kakiuchi S. J. Mol. Cell. Cardiol. 1983; 15: 335-346Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 15Davis B.A. Schwartz A. Samaha F.J. Kranias E.G. J. Biol. Chem. 1983; 258: 13587-13591Abstract Full Text PDF PubMed Google Scholar, 16Jackson W.A. Colyer J. Biochem. J. 1996; 316: 201-207Crossref PubMed Scopus (44) Google Scholar). Some studies have reported additive effects of PKA and CaMKII phosphorylation of PLB on SR Ca2+ transport (13Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (91) Google Scholar, 14Tada M. Inui M. Yamada M. Kadoma M. Kuzuya T. Abe H. Kakiuchi S. J. Mol. Cell. Cardiol. 1983; 15: 335-346Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 17Le Peuch C.J. Haiech J. Demaille J.G. Biochemistry. 1979; 18: 5150-5157Crossref PubMed Scopus (308) Google Scholar, 18Raeymaekers L. Hofmann F. Casteels R. Biochem. J. 1988; 252: 269-273Crossref PubMed Scopus (169) Google Scholar), whereas others (16Jackson W.A. Colyer J. Biochem. J. 1996; 316: 201-207Crossref PubMed Scopus (44) Google Scholar, 19Colyer J. Wang J.H. J. Biol. Chem. 1991; 266: 17486-17493Abstract Full Text PDF PubMed Google Scholar) have proposed that maximal stimulation of the Ca2+ pump occurs by phosphorylation at a single site, and additional phosphorylation of the other site does not further stimulate the pump activity. Several in vivo studies have shown that Ser16phosphorylation or dephosphorylation precedes Thr17phosphorylation or dephosphorylation during exposure or removal of β-agonist stimulation, respectively (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, 12Talosi L. Edes I. Kranias E.G. Am. J. Physiol. 1993; 264: H791-H797PubMed Google Scholar, 20Colyer J. Ann. N. Y. Acad. Sci. 1998; 853: 79-91Crossref PubMed Scopus (73) Google Scholar, 21Napolitano R. Vittone L. Mundina C. Chiappe de Cingolani G. Mattiazzi A. J. Mol. Cell. Cardiol. 1992; 24: 387-396Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 22Calaghan S.C. White E. Colyer J. Pflugers Arch. 1998; 436: 948-956Crossref PubMed Scopus (40) Google Scholar). Furthermore, increases in intracellular Ca2+ to levels higher than those obtained by isoproterenol stimulation, by in the Ca2+ or that stimulation, to in phosphorylation of PLB J.P. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). These results to the that Thr17 phosphorylation has a phosphorylation of Ser16 in vivo. However, other phosphorylation for PLB C. Vittone L. M. de Cingolani Mattiazzi A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. C. M. Mattiazzi A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (44) Google Scholar), indicated that increases in the Ca2+ to the by of protein or phosphorylation of Thr17 and of the and Thus, the on the physiological of Ser16 and Thr17phosphorylation A major in previous studies has been the of expressing wild-type PLB with both phosphorylation which in the relative contribution of each phosphorylation site to cardiac was by the of a PLB in with a to delineate the physiological of each of the phosphorylation Ser16 and in PLB. In a previous a PLB mutant, in which Ser16 was by was in the hearts of the null and studies indicated the lack of Thr17 phosphorylation and attenuation of the β-agonist stimulatory effects in hearts (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). was that phosphorylation of Ser16 be a for Thr17phosphorylation during β-agonist stimulation. that the importance of Ser16 phosphorylation in PLB, the contribution of site relative to Thr17phosphorylation in the stimulatory effects of β-agonists in vivo is not Thus, the study was to the of Ser16 phosphorylation in vivo. A PLB mutant in which Thr17 was by was in the hearts of the null and the were phosphorylation of PLB occur independently of Thr17 in is the relative contribution of Ser16 phosphorylation to the maximal stimulatory effects of and the stimulatory effects of Ser16 and Thr17phosphorylation indicate that phosphorylation of Ser16 in PLB can occur independently of Thr17 in vivo, and phosphorylation is sufficient in mediating the maximal mechanical and stimulatory effects of isoproterenol in cardiac myocytes. PLB phosphorylation mutation of Thr17 to was the PLB by polymerase chain reaction (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, G. Dorn G.W., II Luo W. Harrer J.M. Kadambi V.J. Walsh R.A. Kranias E.G. Circ. Res. 1997; PubMed Scopus Google Scholar). a of PLB and was a with and the was by of The was to a mutant PLB the a with a and a mutant to of the PLB of the and the and were for the to the the mutation in PLB The was and for and site of PLB overexpression which has been to wild-type transgenic mice in (4Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W., II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Crossref PubMed Scopus (276) Google Scholar). The α-myosin heavy chain was to expression of the mutant PLB in the null background. The mutant mice expressing T17A PLB in the PLB mouse were generated in the transgenic mice expressing wild-type or S16A mutant PLB in the null background (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). the expression was of the the PLB with the T17A mutation and the The of the expression was and for The mice the PLB mutant were by to the and the with a of mice with the mutation in the null background were and by of The by the was by of the transgenic mouse hearts. A PLB was a for transgenic mice were in and The cardiac were with of and to gel electrophoresis G. L. Sato Y. Harrer J.M. Kadambi V.J. Hoit B.D. Kranias E.G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and The were with a mouse to phospholamban or SR the with and the were with an of The phospholamban and SR protein were and for the and the were by In of PLB was cardiac mutant (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). PKA phosphorylation of the cardiac was at in of reaction of and of the PKA CaMKII phosphorylation of the cardiac and protein kinase inhibitor were to the reaction were with of a or (CaMKII) which was associated with in PLB. of protein was to and of mouse myocytes was G. L. Sato Y. Harrer J.M. Kadambi V.J. Hoit B.D. Kranias E.G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Chu G. Kranias E.G. Am. J. Physiol. 1998; PubMed Google Scholar). mouse hearts were in a and perfused with at for The and was to the and the The was and through a The was in and and in for further and Ca2+ were the To intracellular Ca2+ were with the of for at and in The myocyte was in a which was on the of an and perfused with at was by a and were and on a A was to myocyte and which the and maximal of and were Ca2+ the were at and by a Ca2+ were the of the The and for of the Ca2+ were To the phosphorylation of PLB in mouse myocytes were transgenic mice obtained mouse hearts with the were and in of the of the myocyte were with a of isoproterenol for The reaction was by and a of of of myocyte by the protein was to each of phosphorylation of PLB, myocyte were to and with PLB that PLB phosphorylated at Ser16 or PLB phosphorylated at Thr17 The was by in with an are was for and of for of were of to to was the of the mouse PLB by (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, G. Dorn G.W., II Luo W. Harrer J.M. Kadambi V.J. Walsh R.A. Kranias E.G. Circ. Res. 1997; PubMed Scopus Google Scholar). The mutation and were by for The was to expression of the PLB mutant in the cardiac compartment of the PLB the PLB were of of PLB that the PLB at whereas the PLB at The a to the a of the PLB The was and the of the mutation of to in PLB was of the which at was detected by of transgenic hearts was not in PLB or wild-type hearts. The wild-type hearts the of the PLB which at and To the PLB protein expression cardiac transgenic mice were and to The protein levels of PLB were in and in the other transgenic with hearts. The expressing PLB, with was for additional expressing similar levels of wild-type or S16A mutant PLB in the hearts of the null background (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) were in parallel. To of the PLB in the SR of PLB and SERCA2 was mouse models with obtained in cardiac the SR protein levels of T17A mutant PLB in the null background were of those of the levels of wild-type or S16A mutant PLB were in (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). significant alterations in SERCA2 protein levels were detected among the PLB hearts with S16A mutant, or T17A mutant PLB in the null background To Ser16 can be phosphorylated in vitro upon mutation of Thr17 to in PLB, cardiac the T17A mutant PLB mice were with the protein kinase A phosphorylation and to and The in PLB was similar in cardiac with wild-type or T17A mutant PLB in the null background The Ca2+-calmodulin-dependent phosphorylation of mutant PLB was in In the of Ca2+ and phosphorylation of wild-type PLB was no in T17A mutant PLB was CaMKII was in the in vitro phosphorylation was to the levels of in the were no significant alterations of CaMKII between S16A mutant, T17A mutant, and hearts Furthermore, phosphorylation of the T17A mutant PLB by protein kinase A was by a PKA inhibitor Ca2+-calmodulin-dependent phosphorylation of the S16A mutant PLB was by a CaMKII inhibitor These the of the PLB phosphorylation and the of the phosphorylation in PLB in vitro phosphorylation of PLB. Cardiac mice with wild-type or mutant PLB in the null background were phosphorylated by the of cAMP-dependent protein kinase or the Ca2+-calmodulin-dependent protein kinase were in the or of PKA inhibitor or CaMKII inhibitor by and to and wild-type PLB in the PLB S16A mutant PLB in the PLB T17A mutant PLB in the PLB PLB To the of T17A mutant PLB on cardiac contractile parameters, myocytes were with and at and of and Ca2+ were PLB hearts exhibited significantly enhanced contractile and Ca2+ kinetics with not with previous W. Wolska B.M. Grupp I.L. Harrer J.M. Haghighi K. Ferguson D.G. Slack J.P. Grupp G. Doetschman T. Solaro R.J. Kranias E.G. Circ. Res. 1996; 78: 839-847Crossref PubMed Scopus (104) Google Scholar, L. Chu G. Kranias E.G. Am. J. Physiol. 1998; PubMed Google Scholar, B.M. Luo W. Kranias E.G. Solaro R.J. Am. J. Physiol. 1996; PubMed Google Scholar, Kranias E.G. J. Physiol. 1997; PubMed Scopus Google Scholar). To the functional of Thr17 phosphorylation in PLB, myocyte mechanics and Ca2+ kinetics were in transgenic hearts expressing similar levels of wild-type PLB in the null background. Furthermore, myocytes hearts expressing similar levels of S16A mutant PLB in the null background were in to the of dual-site PLB phosphorylation. The maximal of and Ca2+ and of Ca2+ were similar among myocytes expressing or T17A mutant PLB in the null background. Thus, the mutant S16A or T17A of PLB myocyte mechanics and Ca2+ to a similar wild-type PLB basal The at the myocyte of the S16A mutant PLB were similar to previous in hearts (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google in myocytes with and at in the or of isoproterenol of to of wild-type PLB in the PLB S16A mutant PLB in the PLB T17A mutant PLB in the PLB hearts. or in the of PLB has been to be a major in the β-adrenergic signaling pathway and a mediator of cardiac responses to β-agonist stimulation (1Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (294) Google Scholar, 2Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar). To the effects of T17A mutant PLB on the myocyte responses to mutant with wild-type or S16A mutant PLB were stimulated at a of and perfused with and stimulation by isoproterenol was associated with significant increases in the and the of and in three The stimulated parameters were similar between T17A and wild-type PLB myocytes, whereas parameters were significantly in S16A mutant PLB In the increases in the Ca2+ and the in the of Ca2+ to of Ca2+ upon maximal isoproterenol stimulation were similar in myocytes expressing wild-type or T17A mutant PLB However, isoproterenol a on the of the and no on the of the in S16A mutant PLB Isoproterenol stimulation was associated with significant phosphorylation of both Ser16 and Thr17 in myocytes expressing wild-type PLB, detected by the phosphorylation to PLB that Ser16 or Thr17 phosphorylation was detectable in wild-type basal In myocytes expressing T17A mutant PLB, of phosphorylated upon isoproterenol stimulation with mutation of Thr17 to there was no phosphorylation of Thr17 detected in were obtained in hearts expressing T17A mutant PLB in the null background not In myocytes expressing S16A mutant PLB, there was no phosphorylation of with previous in hearts (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). isoproterenol stimulation was not associated with detectable phosphorylation of Thr17 in S16A mutant myocytes, in the of Ca2+ and the inhibitor not The of the PLB mouse in with of wild-type or mutant PLB in the null a to the functional of each phosphorylation site in PLB, the between the PKA and CaMKII at the of PLB phosphorylation, and stimulatory effects on cardiac vivo. expression of a PLB mutant, in which of the phosphorylation was to was the α-myosin heavy chain The contractile and Ca2+ kinetics of T17A mutant PLB were characterized in with expressing similar levels of wild-type or S16A mutant PLB (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) in the or of maximal isoproterenol stimulation. of Thr17 to not the of the Ser16 to be phosphorylated by PKA in mutation of Ser16 to not phosphorylation of Thr17 by CaMKII in vitro (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). indicate that Ser16 and Thr17 can be phosphorylated independently in SR with previous in expression and SR (13Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (91) Google Scholar, 14Tada M. Inui M. Yamada M. Kadoma M. Kuzuya T. Abe H. Kakiuchi S. J. Mol. Cell. Cardiol. 1983; 15: 335-346Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 15Davis B.A. Schwartz A. Samaha F.J. Kranias E.G. J. Biol. Chem. 1983; 258: 13587-13591Abstract Full Text PDF PubMed Google Scholar), although study has proposed that phosphorylation of Ser16 by PKA a whereas that of Thr17 by CaMKII in a (16Jackson W.A. Colyer J. Biochem. J. 1996; 316: 201-207Crossref PubMed Scopus (44) Google Scholar). The by which phosphorylation of PLB its effects has been to of the between SERCA2 to and for the removal of of SERCA2 by PLB H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar, Y. K. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. T. Ann. N. Y. Acad. Sci. 1998; 853: PubMed Scopus Google Scholar). The mutant T17A PLB was to the cardiac myocyte function to the wild-type PLB upon its in the null that mutation of to not the of PLB to with and Isoproterenol was associated with increases in the of and of Ca2+ kinetics in T17A mutant PLB Furthermore, the maximal stimulatory effects on mechanics and Ca2+ kinetics in T17A PLB mutant were similar to those in Thus, mutation of T17A in PLB was not associated with significant alterations in the stimulatory effects of PLB in to However, in mutation of to in PLB the stimulatory effects of isoproterenol in with previous in hearts (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These that phosphorylation of Ser16 in PLB be sufficient to the maximal cardiac responses to and the effects of dual-site and PLB phosphorylation not to be vivo. is to that expressing the S16A mutant PLB exhibited increases in the and the maximal of and with upon isoproterenol stimulation, the S16A PLB was not Thus, the functional in S16A mutant myocytes were associated with of PLB phosphorylation, phosphorylation and functional of cardiac the or Ca2+ However, the relative contribution of phosphoproteins in the cardiac responses to β-agonists to be The of β-agonist stimulation on PLB phosphorylation is associated with of the cAMP-dependent signaling of and the of to phosphorylation of Ser16 in PLB of intracellular Ca2+, which is to to phosphorylation of Thr17 in PLB Thus, to between the protein kinase and the of PLB phosphorylation in vivo. The of Ser16 phosphorylation in the β-agonist stimulatory effects in vivo has been proposed by studies (7Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Calaghan S.C. White E. Colyer J. Pflugers Arch. 1998; 436: 948-956Crossref PubMed Scopus (40) Google Scholar, M. E.G. S. Am. J. Physiol. Google Scholar), and a between the of phosphorylation of Ser16 and contractile parameters has been in perfused hearts L. Edes I. Kranias E.G. Am. J. Physiol. 1993; 264: H791-H797PubMed Google Scholar, C. Vittone L. M. de Cingolani Mattiazzi A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and myocytes S.C. White E. Colyer J. Pflugers Arch. 1998; 436: 948-956Crossref PubMed Scopus (40) Google Scholar). However, in Thr17 was and the importance of Ser16 phosphorylation was not The of CaMKII phosphorylation of PLB, of PKA phosphorylation, has been to in vivo. Phosphorylation of PLB by CaMKII was to the of SERCA2 A. L. Am. J. Physiol. 1994; PubMed Google Scholar), and of kinase resulted in of the Ca2+ in cardiac myocytes A. L. Am. J. Physiol. 1994; PubMed Google Scholar, R.A. Mattiazzi A. Am. J. Physiol. 1995; PubMed Google Scholar). CaMKII phosphorylation of PLB has been in the of the cardiac by SR Ca2+ and Ca2+ However, results have been in studies In an study M. Colyer J. Am. J. Physiol. 1997; 273: Google Scholar), no significant in phosphorylation levels of Ser16 of PLB were obtained with stimulation whereas Thr17 phosphorylation was reported to in a and the increases in Thr17 phosphorylation were with enhanced of myocyte and in myocytes M. T. W. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In perfused phosphorylation of upon of protein in the of elevated Ca2+ or C. Vittone L. M. de Cingolani Mattiazzi A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. C. M. Mattiazzi A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Colyer J. Pflugers Arch. 1997; PubMed Scopus Google Scholar), the of Thr17phosphorylation In the study further the of dual-site PLB phosphorylation by the of Ser16 and Thr17phosphorylation in PLB, and the contribution of each phosphorylation site to Ca2+ and contractile parameters in cardiac myocytes in to β-adrenergic stimulation. indicate that Ser16 in PLB can be phosphorylated independently of Thr17 in vitro and in vivo, whereas Thr17 can only be independently phosphorylated in Phosphorylation of Ser16 is sufficient to the maximal cardiac kinetic and contractile responses to β-adrenergic stimulation, that the effects of dual-site and phosphorylation in PLB are not additive in vivo. on we that phosphorylation of Ser16 in PLB a in mediating the cardiac contractile responses to Mattiazzi for and of the