Phosphomimetic Mutations Increase Phospholamban Oligomerization and Alter the Structure of Its Regulatory Complex

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2008 Aug 19

PMID 18708665

Citations 40

Authors

Zhanjia Hou

Eileen M Kelly

Seth L Robia

Affiliations

Soon will be listed here.

Abstract

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K(D)) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET(max) was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.

Citing Articles

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of calcium.

Cleary S, Teng A, Kongmeneck A, Fang X, Phillips T, Cho E J Biol Chem. 2024; 301(2):108118.

PMID: 39710323 PMC: 11791128. DOI: 10.1016/j.jbc.2024.108118.

Phosphorylation of phospholamban promotes SERCA2a activation by dwarf open reading frame (DWORF).

Bovo E, Jamrozik T, Kahn D, Karkut P, Robia S, Zima A Cell Calcium. 2024; 121:102910.

PMID: 38823350 PMC: 11247691. DOI: 10.1016/j.ceca.2024.102910.

Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation.

Funk F, Kronenbitter A, Hackert K, Oebbeke M, Klebe G, Barth M Cardiovasc Res. 2023; 119(7):1568-1582.

PMID: 36869774 PMC: 10318394. DOI: 10.1093/cvr/cvad037.

Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.

Phillips T, Hauck G, Pribadi M, Cho E, Cleary S, Robia S Biophys J. 2022; 122(2):301-309.

PMID: 36523160 PMC: 9892615. DOI: 10.1016/j.bpj.2022.12.014.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

Seflova J, Habibi N, Yap J, Cleary S, Fang X, Kekenes-Huskey P J Biol Chem. 2022; 298(5):101865.

PMID: 35339486 PMC: 9048134. DOI: 10.1016/j.jbc.2022.101865.

References

Oxenoid K, Rice A, Chou J . Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo-phosphorylated states. Protein Sci. 2007; 16(9):1977-83. PMC: 2206959. DOI: 10.1110/ps.072975107. View

Patterson G, Piston D, Barisas B . Förster distances between green fluorescent protein pairs. Anal Biochem. 2000; 284(2):438-40. DOI: 10.1006/abio.2000.4708. View

Oxenoid K, Chou J . The structure of phospholamban pentamer reveals a channel-like architecture in membranes. Proc Natl Acad Sci U S A. 2005; 102(31):10870-5. PMC: 1182456. DOI: 10.1073/pnas.0504920102. View

Chen Z, Akin B, Jones L . Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12. J Biol Chem. 2007; 282(29):20968-76. DOI: 10.1074/jbc.M703516200. View

Mahaney J, Albers R, Waggoner J, Kutchai H, Froehlich J . Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition. Biochemistry. 2005; 44(21):7713-24. DOI: 10.1021/bi048011i. View

Toyoshima C, Asahi M, Sugita Y, Khanna R, Tsuda T, MacLennan D . Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase. Proc Natl Acad Sci U S A. 2003; 100(2):467-72. PMC: 141018. DOI: 10.1073/pnas.0237326100. View

Kimura Y, Kurzydlowski K, Tada M, Maclennan D . Phospholamban inhibitory function is activated by depolymerization. J Biol Chem. 1997; 272(24):15061-4. DOI: 10.1074/jbc.272.24.15061. View

Autry J, Jones L . Functional Co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation. J Biol Chem. 1997; 272(25):15872-80. DOI: 10.1074/jbc.272.25.15872. View

Wegener A, Simmerman H, Lindemann J, Jones L . Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation. J Biol Chem. 1989; 264(19):11468-74. View

10.

Asahi M, McKenna E, Kurzydlowski K, Tada M, Maclennan D . Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP. J Biol Chem. 2000; 275(20):15034-8. DOI: 10.1074/jbc.275.20.15034. View

11.

Brittsan A, Carr A, Schmidt A, Kranias E . Maximal inhibition of SERCA2 Ca(2+) affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban. J Biol Chem. 2000; 275(16):12129-35. DOI: 10.1074/jbc.275.16.12129. View

12.

Hutter M, Krebs J, Meiler J, Griesinger C, Carafoli E, Helms V . A structural model of the complex formed by phospholamban and the calcium pump of sarcoplasmic reticulum obtained by molecular mechanics. Chembiochem. 2002; 3(12):1200-8. DOI: 10.1002/1439-7633(20021202)3:12<1200::AID-CBIC1200>3.0.CO;2-H. View

13.

Smith S, Kawakami T, Liu W, Ziliox M, Aimoto S . Helical structure of phospholamban in membrane bilayers. J Mol Biol. 2001; 313(5):1139-48. DOI: 10.1006/jmbi.2001.5101. View

14.

Karim C, Zhang Z, Howard E, Torgersen K, Thomas D . Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA. J Mol Biol. 2006; 358(4):1032-40. DOI: 10.1016/j.jmb.2006.02.051. View

15.

Zamoon J, Mascioni A, Thomas D, Veglia G . NMR solution structure and topological orientation of monomeric phospholamban in dodecylphosphocholine micelles. Biophys J. 2003; 85(4):2589-98. PMC: 1303482. DOI: 10.1016/S0006-3495(03)74681-5. View

16.

Clayton J, Hughes E, Middleton D . The cytoplasmic domains of phospholamban and phospholemman associate with phospholipid membrane surfaces. Biochemistry. 2005; 44(51):17016-26. DOI: 10.1021/bi0511383. View

17.

Dolinsky T, Nielsen J, McCammon J, Baker N . PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004; 32(Web Server issue):W665-7. PMC: 441519. DOI: 10.1093/nar/gkh381. View

18.

Li J, Bigelow D, Squier T . Conformational changes within the cytosolic portion of phospholamban upon release of Ca-ATPase inhibition. Biochemistry. 2004; 43(13):3870-9. DOI: 10.1021/bi036183u. View

19.

Traaseth N, Ha K, Verardi R, Shi L, Buffy J, Masterson L . Structural and dynamic basis of phospholamban and sarcolipin inhibition of Ca(2+)-ATPase. Biochemistry. 2007; 47(1):3-13. PMC: 2699759. DOI: 10.1021/bi701668v. View

20.

Hoshijima M, Ikeda Y, Iwanaga Y, Minamisawa S, Date M, Gu Y . Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat Med. 2002; 8(8):864-71. DOI: 10.1038/nm739. View