Abstract
Phospholamban (PLB), the regulator of the cardiac sarcoplasmic reticulum (SR) Ca2+ pump is specifically phosphorylated at Ser16 and Thr17 by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaMK), respectively. The regulation of this dual-site phosphorylation of amino acid residues in direct proximity is only poorly understood. In order to study the site-specific phosphorylation of PLB, we used a synthetic peptide (PLB-24) corresponding to the cytosolic part of the PLB monomer with the phosphorylation sites as a model substrate. PLB-24 possesses substrate properties as the native PLB as demonstrated by phosphorylation with exogenous, purified PKA, cGMP-dependent protein kinase (PKG) and a type II CaMK (CaMKII). In isolated vesicles of crdiac SR there was a rapid phosphorylation of the peptide by the endogenous PKA (SR-PKA) and CaMK (SR-CaMK), but not under conditions that activate PKG. Both SR-PKA and SR-CaMK incorporated the same amount of32P into PLB-24, 0.60±0.01 nmol32P/mg SR protein and 0.61±0.03 nmol32P/mg SR protein, respectively. Phosphorylation by SR-PKA was abolished by the specific PKA inhibitor (IC50=0.2μM), whereas SR-CaMK phosphorylation was inhibited by calmidazolium (IC50=1.6μM) and a CaMKII-specific inhibitor peptide (IC50=2.5μM). Phosphorylation by SR-PKA was exclusively at Ser, whereas SR-CaMK phosphorylated only Thr. After simultaneous activation of both SR-kinases32P incorporation into PLB-24 was additive and occurred at Ser as well as at Thr. Sequential activation of SR-PKA and SR-CaMK also caused the additive phosphorylation of PLB-24 independently of which kinase was activated first. Thus, at the monomeric level of PLB the respective phosphorylation site appears to be accessible to its related SR protein kinasein vitro even when the adjacent site is phosphorylated.
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References
Baltas I, Karczewski P, Krause E-G (1995) The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ-CaM kinase isozyme. FEBS Lett 373: 71–75
Bilezikjian LM, Kranias EG, Potter JD, Schwartz A (1981) Studies on phosphorylation of canine cardiac sarcoplasmic reticulum by calmodulin-dependent protein kinase. Circ Res 49: 1356–1362
Colyer J, Wang JH (1991) Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban. J Biol Chem 266: 17486–17493
Cornwell TL, Pryzwanski KB, Wyatt TA, Lincoln TM (1991) Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 40: 923–931
Harigaya S, Schwartz A (1969) Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. Circ Res 25: 781–794
Hohenegger M, Suko J (1993) Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases. Biochem J 296: 303–308
Hubbard JA, MacLachlan LK, Meenan E, Salter CJ, Reid DG, Lahouratate P, Humphries J, Stevens N, Bell D, Neville WA, Murray KJ, Darker JG (1994) Confirmation of the cytosolic domain of phospholamban by NMR and CD. Mol Membr Biol 11: 263–269
Hughes G, East JM, Lee AG (1994) The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca2+-ATPase. Biochem J 303: 511–516
Inui JM, Kimura Y, Sasaki T, Tada M (1990) Molecular mechanisms of calcium uptake and release by sarcoplasmic reticulum. Japan Circ J 54: 1185–1191
Jett M-F, Schworer CM, Bass M, Soderling TR (1987) Identification of membrane-bound calcium, calmodulin-dependent protein kinase II in canine heart. Arch Biochem Biophys 255: 354–360
Karczewski P, Bartel S, Haase H, Krause E-G (1987) Isoproterenol induces both cAMP- and calcium-dependent phosphorylation of phospholamban in canine heart in vivo. Biomed Biochim Acta 46: 433–439
Kim HW, Steenart NAE, Ferguson DG, Kranias EG (1990) Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2+-ATPase with phospholamban in phospholipid vesicles. J Biol Chem 265: 1702–1709
Kranias EG, Bilezikjian LM, Potter JD, Piascik MT, Schwartz A (1980) The role of calmodulin in regulation of cardiac sarcoplasmic reticulum phosphorylation. Ann NY Acad Sci 356: 279–290
LePeuch CJ, Guilleaux J, DeMaille JG (1980) Phospholamban phosphorylation in the perfused rat heart is not solely dependent on β-adrenergic stimulation. FEBS Lett 114: 165–168
LePeuch CJ, Haiech J, Demaille JG (1979) Concerted regulation of cardiac sarcoplasmic reticulum calcium transport by cyclic adenosine monophosphate-dependent and calcium-calmodulin-dependent phosphorylations. Biochemistry 18: 5150–5157
Lindemann JP, Watanabe AM (1985) Phosphorylation of phospholamban in the intact myocardium. J Biol Chem 260: 4516–4525
Lippmann C, Lindschau C, Erdmann VA (1992) Thin-layer electrophoresis with PhastSystem facilitates analysis of phosphoamino acids from proteins bound to Immobilon. Electrophoresis 13: 666–668
Louis CF, Mafitt M, Jarvis B (1982) Factors that modify the molecular size of phospholamban, the 23,000-dalton cardiac sarcoplasmic reticulum phosphoprotein. J Biol Chem 257: 15182–15186
Luo W, Grupp I, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, Kranias EG (1994) Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of β-adrenergic stimulation. Circ Res 75: 401–409
Mery P-F, Lohmann SM, Walter U, Fischmeister R (1991) Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci USA 88: 1197–1201
Mortishiresmith RJ, Pitzenberger SM, Burke CJ, Middaugh CR, Garsky VM, Johnson RG (1995) Solution structure of the cytoplasmic domain of phospholamban-phosphorylation leads to a local perturbation in secondary structure. Biochemistry 34: 7603–7613
Raeymaekers L, Hofmann F, Casteels R (1988) Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle. Biochem J 252: 269–273
Peters KA, Demaille JQ, Fischer EH (1977) Adenosine 3′∶5′-monophosphate dependent protein kinase from bovine heart. Characterization of the catalytic subunit. Biochemistry 26: 5691–5697
Sasaki T, Inui M, Kimura Y, Kuzuya T, Tada M (1992) Molecular mechanisms of regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum. J Biol Chem 267: 1674–1679
Simmerman HKB, Collins JH, Theibert JL, Wegener AD, Jones LR (1986) Sequence analysis of phospholamban: identification of phosphorylation sites and two major structural domains. J Biol Chem 261: 13333–13341
Tada M, Inui M, Yamada M, Kadoma MA, Kuzuya T, Abe H, Kakiuchi S (1983) Effect of phospholamban phosphorylation catalyzed by adenosine 3′∶5′-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum. J Mol Cell Cardiol 15: 335–346
Tada M, Katz A (1982) Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol 44: 401–423
Vorherr T, Chiesi M, Schwaller R, Carafoli E (1992) Regulation of the calcium ion pump of sarcoplasmic reticulum: Reversible inhibition by phospholamban and by the calmodulin binding domain of the plasma membrane calcium ion pump. Biochemistry 31: 371–376
Walter U, Miller P, Wilson F, Menkes D, Greengard P (1980) Immunological distinction between guanosine 3′∶5′-monophosphate-dependent protein kinases. J Biol Chem 255: 3757–3762
Wegener AD, Simmerman HKB, Liepniekes J, Jones LR (1986) Proteolytic cleavage of phospholamban purified from canine sarcoplasmic reticulum vesicles: generation of a low resolution model of phospholamban structure. J Biol Chem 261: 5154–5159
Wegener AD, Simmerman HKB, Lindemann JP, Jones LR (1989) Phospholamban phosphorylation in intact ventricles. J Biol Chem 264: 11486–11474
Xu A, Hawkins C, Narayanan N (1995) Phosphorylation and activation of the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum by Ca2+/calmodulin-dependent protein kinase. J Biol Chem 268: 8394–8397
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Karczewski, P., Kuschel, M., Baltas, L.G. et al. Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide- and Ca2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum. Basic Res Cardiol 92 (Suppl 1), 37–43 (1997). https://doi.org/10.1007/BF00794066
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DOI: https://doi.org/10.1007/BF00794066