Phosphorylation of Ser16 is a prerequisite for Thr17 phosphorylation in phospholamban, and preventing phosphoserine formation attenuates β-agonist stimulatory responses in the mammalian heart.
Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 → Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the β-agonist stimulatory responses in the mammalian heart. Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 → Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the β-agonist stimulatory responses in the mammalian heart. Phospholamban (PLB) 1The abbreviations used are: PLB, phospholamban; SR, sarcoplasmic reticulum; PCR, polymerase chain reaction; kb, kilobase(s); MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase. 1The abbreviations used are: PLB, phospholamban; SR, sarcoplasmic reticulum; PCR, polymerase chain reaction; kb, kilobase(s); MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase. is a regulator of the affinity of the cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase for Ca2+. Dephosphorylated PLB is an inhibitor, and phosphorylation of PLB removes its inhibitory effects on the SR Ca2+-ATPase. Recently, the critical role of PLB in the regulation of cardiac contractility has been defined through gene transfer (1Kadambi 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 (275) Google Scholar) and knockout (2Luo 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 (627) Google Scholar) technology in the mouse. Cardiac-specific overexpression of PLB was associated with decreases in the affinity of the SR Ca2+-ATPase for Ca2+ and depressed cardiac function, whereas PLB deficiency resulted in increased Ca2+ affinity of the Ca2+-ATPase and enhanced myocardial performance. Furthermore, the stimulatory effects to β-adrenergic agonists were more pronounced in the PLB-overexpressing hearts, whereas these effects were attenuated in the PLB-knockout hearts compared with wild types (1Kadambi 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 (275) Google Scholar, 2Luo 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 (627) Google Scholar). These studies suggested that PLB plays a prominent role in the heart's responses to β-agonists. However, PLB is phosphorylated on both Ser16and Thr17 during isoproterenol stimulation (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar) and the relative contribution of each site in the altered contractile responses of the heart is not presently well known. In vitro studies have shown that Ser16 is phosphorylated by cAMP-dependent protein kinase, whereas Thr17 is phosphorylated by Ca2+-calmodulin-dependent protein kinase (4Simmerman 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). Phosphorylation of each site occurs in an independent manner, although it is not presently clear whether the stimulatory effects of the two phosphorylations on SR Ca2+transport are additive (5Bilezikjian L.M. Kranias E.G. Potter J.D. Schwartz A. Circ. Res. 1981; 49: 1356-1362Crossref PubMed Scopus (60) Google Scholar, 6Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (89) Google Scholar, 7Raeymaekers L. Hofmann F. Casteels R. Biochem. J. 1988; 252: 269-273Crossref PubMed Scopus (169) Google Scholar, 8Tada 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 (101) Google Scholar, J. J.H. J. Biol. Chem. Full Text PDF PubMed Google vivo studies have shown that of PLB by Ca2+-calmodulin-dependent protein kinase has as a prerequisite the by cAMP-dependent protein kinase J.P. Jones L.R. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar, J.P. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar, L. G. A. J. PubMed Google Scholar, R. L. G. A. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google Scholar, L. G. A. Mol. Cell. Biochem. PubMed Scopus Google Scholar, A. L. J. 1994; PubMed Google Scholar). Furthermore, of to levels by resulted in that during β-adrenergic to PLB (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, J.P. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). However, a phosphorylation for PLB, indicated that cAMP-dependent and Ca2+-calmodulin-dependent phosphorylation of PLB in an independent and effects additive in vivo L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the role of dual site phosphorylation of PLB is not of the PLB knockout mouse in with technology have with an to the the cAMP-dependent and the Ca2+-calmodulin-dependent of PLB phosphorylation in the regulation of basal and β-agonist cardiac contractility in The of the were determine whether of PLB in the is and whether it is to the hyperdynamic cardiac of the PLB knockout and the physiological role of Thr17 phosphorylation in PLB in the of Ser16 → by of mutant PLB in the knockout results are the to that a cardiac generated by gene reversed by reinsertion of the gene in the In we that of PLB resulted in enhanced basal cardiac contractile parameters assessed the and levels (2Luo 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 (627) Google Scholar, G. W. J.P. M. Grupp I.L. Kranias E.G. Circ. Res. 1996; PubMed Scopus Google Scholar, W. Kranias E.G. J. 1996; PubMed Google Scholar, B.D. Kranias E.G. Walsh R.A. Circ. Res. PubMed Scopus Google Scholar). In we used the heavy chain to of wild-type PLB in the knockout and of the hyperdynamic function of the hearts. The of of the contractile parameters was to the levels of PLB, in with studies in and (2Luo 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 (627) Google Scholar, W. Grupp I.L. Harrer J.M. J.P. Grupp G. Doetschman T. Kranias E.G. Circ. Res. 1996; PubMed Scopus Google Scholar). The of PLB in the altered by of the knockout indicated that the mouse an for of PLB in the heart and of in the mutant we we to the significance of dual site PLB phosphorylation during β-adrenergic stimulation. was the PLB Ser16 to Ala, and mutant PLB was in the knockout of cardiac and that the mutant PLB was in the SR Transgenic expressing similar levels of wild-type or mutant PLB in the similar contractile parameters basal that the mutant PLB was of contractility in a similar to that for wild-type However, isoproterenol stimulation was associated with much lower of the of and in the hearts compared with hearts, whereas the heart responses were similar these is to that the in contractile parameters of the hearts were similar to of PLB-knockout hearts β-adrenergic stimulation. These findings that of Ser16 in PLB the contribution of in the stimulatory responses of the heart to was in the was 32P labeling of mutant PLB isoproterenol stimulation. However, in the same hearts, the of phosphorylation of and in the was similar hearts expressing mutant or wild-type cardiac PLB were for the attenuated responses of the hearts. To the that the lack of in due to of the Ser16 to Ala, cardiac and were vitro phosphorylation for the Ca2+-calmodulin-dependent protein kinase. The of mutant PLB phosphorylation was similar to that of wild-type PLB and formation was the phospholamban phosphorylation that the mutant PLB form was of phosphorylated on role of PLB phosphorylation by cAMP-dependent and Ca2+-calmodulin-dependent protein has been the of by L. Hofmann F. Casteels R. Biochem. J. 1988; 252: 269-273Crossref PubMed Scopus (169) Google 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 (101) Google and Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (89) Google Scholar) indicated that the stimulatory effects of the two protein on sarcoplasmic reticulum Ca2+ whereas a by and J. J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) suggested that stimulation of the Ca2+ occurs by PLB phosphorylation a site and that phosphorylation of the site not Furthermore, the in that phosphorylation of PLB by cAMP-dependent and Ca2+-calmodulin-dependent protein occurs in an independent manner, whereas in vivo findings (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, R. L. G. A. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google Scholar, L. Kranias E.G. J. 264: Google Scholar) indicate that of Thr17 occurs to of Ser16 during β-adrenergic stimulation. findings in transgenic Thr17 in PLB phosphorylated in the of Ser16 phosphorylation, isoproterenol stimulation of and phosphorylation of Thr17 in PLB not phosphorylation of Ser16 in in vitro experiments. phosphorylation of of Ser16 phosphorylation in whereas phosphorylation of the to a prerequisite for in formation during β-agonist stimulation. in vivo and in due to in the levels of to the SR Ca2+-calmodulin-dependent protein kinase. In vitro However, in in vivo phosphorylation of Ser16 to and the SR Ca2+ SR Ca2+ to increased by the SR, of the Ca2+-calmodulin-dependent protein kinase, and phosphorylation of Thr17 in The phosphorylation and of the Ca2+ to the increased Ca2+ levels vivo phosphorylation of Thr17 in PLB L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of the protein was suggested to an for Thr17 phosphorylation L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google not to role in transgenic isoproterenol significance of Ca2+-calmodulin-dependent phosphorylation of PLB has been in cardiac A. L. J. 1994; PubMed Google Scholar, R.A. A. J. PubMed Google Scholar). Phosphorylation of PLB by Ca2+-calmodulin-dependent protein kinase II was suggested to the of the SR whereas phosphorylation by protein kinase increased the Ca2+ affinity of the A. L. J. 1994; PubMed Google Scholar). Furthermore, of Ca2+-calmodulin-dependent protein kinase II was shown to sarcoplasmic reticulum Ca2+ and the of of protein kinase R.A. A. J. PubMed Google that the in PLB in regulation of Ca2+ and prevention of Ca2+ L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. 1996; PubMed Google Scholar, J. PubMed Scopus Google findings indicate that the hyperdynamic PLB knockout reversed by of PLB in the and the of technology in PLB studies in of mutant PLB in Ser16 was by Ala in the knockout indicated that the phosphorylation of Thr17 in PLB phosphorylation of Ser16 during β-adrenergic stimulation. In the of Ser16 phosphorylation, the of the stimulatory effects by was similar to that in PLB knockout hearts, that cardiac PLB these studies transgenic the Thr17 → Ala or → in PLB the of dual site phosphorylation in PLB and the of each phosphorylation site physiological and Phospholamban (PLB) 1The abbreviations used are: PLB, phospholamban; SR, sarcoplasmic reticulum; PCR, polymerase chain reaction; kb, kilobase(s); MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase. 1The abbreviations used are: PLB, phospholamban; SR, sarcoplasmic reticulum; PCR, polymerase chain reaction; kb, kilobase(s); MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase. is a regulator of the affinity of the cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase for Ca2+. Dephosphorylated PLB is an inhibitor, and phosphorylation of PLB removes its inhibitory effects on the SR Ca2+-ATPase. Recently, the critical role of PLB in the regulation of cardiac contractility has been defined through gene transfer (1Kadambi 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 (275) Google Scholar) and knockout (2Luo 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 (627) Google Scholar) technology in the mouse. Cardiac-specific overexpression of PLB was associated with decreases in the affinity of the SR Ca2+-ATPase for Ca2+ and depressed cardiac function, whereas PLB deficiency resulted in increased Ca2+ affinity of the Ca2+-ATPase and enhanced myocardial performance. Furthermore, the stimulatory effects to β-adrenergic agonists were more pronounced in the PLB-overexpressing hearts, whereas these effects were attenuated in the PLB-knockout hearts compared with wild types (1Kadambi 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 (275) Google Scholar, 2Luo 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 (627) Google Scholar). These studies suggested that PLB plays a prominent role in the heart's responses to β-agonists. However, PLB is phosphorylated on both Ser16and Thr17 during isoproterenol stimulation (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar) and the relative contribution of each site in the altered contractile responses of the heart is not presently well known. In vitro studies have shown that Ser16 is phosphorylated by cAMP-dependent protein kinase, whereas Thr17 is phosphorylated by Ca2+-calmodulin-dependent protein kinase (4Simmerman 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). Phosphorylation of each site occurs in an independent manner, although it is not presently clear whether the stimulatory effects of the two phosphorylations on SR Ca2+transport are additive (5Bilezikjian L.M. Kranias E.G. Potter J.D. Schwartz A. Circ. Res. 1981; 49: 1356-1362Crossref PubMed Scopus (60) Google Scholar, 6Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (89) Google Scholar, 7Raeymaekers L. Hofmann F. Casteels R. Biochem. J. 1988; 252: 269-273Crossref PubMed Scopus (169) Google Scholar, 8Tada 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 (101) Google Scholar, J. J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In vivo studies have shown that of PLB by Ca2+-calmodulin-dependent protein kinase has as a prerequisite the by cAMP-dependent protein kinase J.P. Jones L.R. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar, J.P. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar, L. G. A. J. PubMed Google Scholar, R. L. G. A. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google Scholar, L. G. A. Mol. Cell. Biochem. PubMed Scopus Google Scholar, A. L. J. 1994; PubMed Google Scholar). Furthermore, of to levels by resulted in that during β-adrenergic to PLB (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, J.P. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). However, a phosphorylation for PLB, indicated that cAMP-dependent and Ca2+-calmodulin-dependent phosphorylation of PLB in an independent and effects additive in vivo L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the role of dual site phosphorylation of PLB is not The of the PLB knockout mouse in with technology have with an to the the cAMP-dependent and the Ca2+-calmodulin-dependent of PLB phosphorylation in the regulation of basal and β-agonist cardiac contractility in The of the were determine whether of PLB in the is and whether it is to the hyperdynamic cardiac of the PLB knockout and the physiological role of Thr17 phosphorylation in PLB in the of Ser16 → by of mutant PLB in the knockout results are the to that a cardiac generated by gene reversed by reinsertion of the gene in the In we that of PLB resulted in enhanced basal cardiac contractile parameters assessed the and levels (2Luo 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 (627) Google Scholar, G. W. J.P. M. Grupp I.L. Kranias E.G. Circ. Res. 1996; PubMed Scopus Google Scholar, W. Kranias E.G. J. 1996; PubMed Google Scholar, B.D. Kranias E.G. Walsh R.A. Circ. Res. PubMed Scopus Google Scholar). In we used the heavy chain to of wild-type PLB in the knockout and of the hyperdynamic function of the hearts. The of of the contractile parameters was to the levels of PLB, in with studies in and (2Luo 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 (627) Google Scholar, W. Grupp I.L. Harrer J.M. J.P. Grupp G. Doetschman T. Kranias E.G. Circ. Res. 1996; PubMed Scopus Google Scholar). The of PLB in the altered by of the knockout indicated that the mouse an for of PLB in the heart and of in the mutant we we to the significance of dual site PLB phosphorylation during β-adrenergic stimulation. was the PLB Ser16 to Ala, and mutant PLB was in the knockout of cardiac and that the mutant PLB was in the SR Transgenic expressing similar levels of wild-type or mutant PLB in the similar contractile parameters basal that the mutant PLB was of contractility in a similar to that for wild-type However, isoproterenol stimulation was associated with much lower of the of and in the hearts compared with hearts, whereas the heart responses were similar these is to that the in contractile parameters of the hearts were similar to of PLB-knockout hearts β-adrenergic stimulation. These findings that of Ser16 in PLB the contribution of in the stimulatory responses of the heart to was in the was 32P labeling of mutant PLB isoproterenol stimulation. However, in the same hearts, the of phosphorylation of and in the was similar hearts expressing mutant or wild-type cardiac PLB were for the attenuated responses of the hearts. To the that the lack of in due to of the Ser16 to Ala, cardiac and were vitro phosphorylation for the Ca2+-calmodulin-dependent protein kinase. The of mutant PLB phosphorylation was similar to that of wild-type PLB and formation was the phospholamban phosphorylation that the mutant PLB form was of phosphorylated on role of PLB phosphorylation by cAMP-dependent and Ca2+-calmodulin-dependent protein has been the of by L. Hofmann F. Casteels R. Biochem. J. 1988; 252: 269-273Crossref PubMed Scopus (169) Google 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 (101) Google and Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (89) Google Scholar) indicated that the stimulatory effects of the two protein on sarcoplasmic reticulum Ca2+ whereas a by and J. J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) suggested that stimulation of the Ca2+ occurs by PLB phosphorylation a site and that phosphorylation of the site not Furthermore, the in that phosphorylation of PLB by cAMP-dependent and Ca2+-calmodulin-dependent protein occurs in an independent manner, whereas in vivo findings (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, R. L. G. A. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google Scholar, L. Kranias E.G. J. 264: Google Scholar) indicate that of Thr17 occurs to of Ser16 during β-adrenergic stimulation. findings in transgenic Thr17 in PLB phosphorylated in the of Ser16 phosphorylation, isoproterenol stimulation of and phosphorylation of Thr17 in PLB not phosphorylation of Ser16 in in vitro experiments. phosphorylation of of Ser16 phosphorylation in whereas phosphorylation of the to a prerequisite for in formation during β-agonist stimulation. in vivo and in due to in the levels of to the SR Ca2+-calmodulin-dependent protein kinase. In vitro However, in in vivo phosphorylation of Ser16 to and the SR Ca2+ SR Ca2+ to increased by the SR, of the Ca2+-calmodulin-dependent protein kinase, and phosphorylation of Thr17 in The phosphorylation and of the Ca2+ to the increased Ca2+ levels vivo phosphorylation of Thr17 in PLB L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of the protein was suggested to an for Thr17 phosphorylation L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google not to role in transgenic isoproterenol significance of Ca2+-calmodulin-dependent phosphorylation of PLB has been in cardiac A. L. J. 1994; PubMed Google Scholar, R.A. A. J. PubMed Google Scholar). Phosphorylation of PLB by Ca2+-calmodulin-dependent protein kinase II was suggested to the of the SR whereas phosphorylation by protein kinase increased the Ca2+ affinity of the A. L. J. 1994; PubMed Google Scholar). Furthermore, of Ca2+-calmodulin-dependent protein kinase II was shown to sarcoplasmic reticulum Ca2+ and the of of protein kinase R.A. A. J. PubMed Google that the in PLB in regulation of Ca2+ and prevention of Ca2+ L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. 1996; PubMed Google Scholar, J. PubMed Scopus Google findings indicate that the hyperdynamic PLB knockout reversed by of PLB in the and the of technology in PLB studies in of mutant PLB in Ser16 was by Ala in the knockout indicated that the phosphorylation of Thr17 in PLB phosphorylation of Ser16 during β-adrenergic stimulation. In the of Ser16 phosphorylation, the of the stimulatory effects by was similar to that in PLB knockout hearts, that cardiac PLB these studies transgenic the Thr17 → Ala or → in PLB the of dual site phosphorylation in PLB and the of each phosphorylation site physiological and results are the to that a cardiac generated by gene reversed by reinsertion of the gene in the In we that of PLB resulted in enhanced basal cardiac contractile parameters assessed the and levels (2Luo 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 (627) Google Scholar, G. W. J.P. M. Grupp I.L. Kranias E.G. Circ. Res. 1996; PubMed Scopus Google Scholar, W. Kranias E.G. J. 1996; PubMed Google Scholar, B.D. Kranias E.G. Walsh R.A. Circ. Res. PubMed Scopus Google Scholar). In we used the heavy chain to of wild-type PLB in the knockout and of the hyperdynamic function of the hearts. The of of the contractile parameters was to the levels of PLB, in with studies in and (2Luo 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 (627) Google Scholar, W. Grupp I.L. Harrer J.M. J.P. Grupp G. Doetschman T. Kranias E.G. Circ. Res. 1996; PubMed Scopus Google Scholar). The of PLB in the altered by of the knockout indicated that the mouse an for of PLB in the heart and of in the mutant we we to the significance of dual site PLB phosphorylation during β-adrenergic stimulation. was the PLB Ser16 to Ala, and mutant PLB was in the knockout of cardiac and that the mutant PLB was in the SR Transgenic expressing similar levels of wild-type or mutant PLB in the similar contractile parameters basal that the mutant PLB was of contractility in a similar to that for wild-type However, isoproterenol stimulation was associated with much lower of the of and in the hearts compared with hearts, whereas the heart responses were similar these is to that the in contractile parameters of the hearts were similar to of PLB-knockout hearts β-adrenergic stimulation. These findings that of Ser16 in PLB the contribution of in the stimulatory responses of the heart to was in the was 32P labeling of mutant PLB isoproterenol stimulation. However, in the same hearts, the of phosphorylation of and in the was similar hearts expressing mutant or wild-type cardiac PLB were for the attenuated responses of the hearts. To the that the lack of in due to of the Ser16 to Ala, cardiac and were vitro phosphorylation for the Ca2+-calmodulin-dependent protein kinase. The of mutant PLB phosphorylation was similar to that of wild-type PLB and formation was the phospholamban phosphorylation that the mutant PLB form was of phosphorylated on The role of PLB phosphorylation by cAMP-dependent and Ca2+-calmodulin-dependent protein has been the of by L. Hofmann F. Casteels R. Biochem. J. 1988; 252: 269-273Crossref PubMed Scopus (169) Google 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 (101) Google and Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (89) Google Scholar) indicated that the stimulatory effects of the two protein on sarcoplasmic reticulum Ca2+ whereas a by and J. J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) suggested that stimulation of the Ca2+ occurs by PLB phosphorylation a site and that phosphorylation of the site not Furthermore, the in that phosphorylation of PLB by cAMP-dependent and Ca2+-calmodulin-dependent protein occurs in an independent manner, whereas in vivo findings (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, R. L. G. A. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google Scholar, L. Kranias E.G. J. 264: Google Scholar) indicate that of Thr17 occurs to of Ser16 during β-adrenergic stimulation. findings in transgenic Thr17 in PLB phosphorylated in the of Ser16 phosphorylation, isoproterenol stimulation of and phosphorylation of Thr17 in PLB not phosphorylation of Ser16 in in vitro experiments. phosphorylation of of Ser16 phosphorylation in whereas phosphorylation of the to a prerequisite for in formation during β-agonist stimulation. in vivo and in due to in the levels of to the SR Ca2+-calmodulin-dependent protein kinase. In vitro However, in in vivo phosphorylation of Ser16 to and the SR Ca2+ SR Ca2+ to increased by the SR, of the Ca2+-calmodulin-dependent protein kinase, and phosphorylation of Thr17 in The phosphorylation and of the Ca2+ to the increased Ca2+ levels vivo phosphorylation of Thr17 in PLB L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of the protein was suggested to an for Thr17 phosphorylation L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google not to role in transgenic isoproterenol stimulation. The significance of Ca2+-calmodulin-dependent phosphorylation of PLB has been in cardiac A. L. J. 1994; PubMed Google Scholar, R.A. A. J. PubMed Google Scholar). Phosphorylation of PLB by Ca2+-calmodulin-dependent protein kinase II was suggested to the of the SR whereas phosphorylation by protein kinase increased the Ca2+ affinity of the A. L. J. 1994; PubMed Google Scholar). Furthermore, of Ca2+-calmodulin-dependent protein kinase II was shown to sarcoplasmic reticulum Ca2+ and the of of protein kinase R.A. A. J. PubMed Google that the in PLB in regulation of Ca2+ and prevention of Ca2+ L. M. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. 1996; PubMed Google Scholar, J. PubMed Scopus Google Scholar). In findings indicate that the hyperdynamic PLB knockout reversed by of PLB in the and the of technology in PLB studies in of mutant PLB in Ser16 was by Ala in the knockout indicated that the phosphorylation of Thr17 in PLB phosphorylation of Ser16 during β-adrenergic stimulation. In the of Ser16 phosphorylation, the of the stimulatory effects by was similar to that in PLB knockout hearts, that cardiac PLB these studies transgenic the Thr17 → Ala or → in PLB the of dual site phosphorylation in PLB and the of each phosphorylation site physiological and J. for the heavy chain and J. for of the transgenic
Luo et al. (Sun,) studied this question.