CAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban

Larry R. Masterson, Tao Yu, Lei Shi, Yi Wang, Martin Gustavsson, Michael M. Mueller, Gianluigi Veglia

Research output: Contribution to journalArticlepeer-review

52 Scopus citations


Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca2+-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.

Original languageEnglish (US)
Pages (from-to)155-164
Number of pages10
JournalJournal of Molecular Biology
Issue number2
StatePublished - Sep 16 2011

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health Grants GM72701 to G.V. and T32DE007288 to L.R.M. and predoctoral fellowship 10PRE3860050 from the Midwest Affiliate American Heart Association to M.G. NMR instrumentation was at the University of Minnesota High Field NMR Center (National Science Foundation BIR-961477 ), University of Minnesota Medical School. Modeling and calculations were carried out at the Minnesota Supercomputing Institute.


  • NMR
  • PKA
  • conformational selection
  • phosphorylation kinetics


Dive into the research topics of 'CAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban'. Together they form a unique fingerprint.

Cite this