title: cAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban.
abstract: 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 Ca(2+)-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.
authors: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors: Larry R
authors: LR
authors: Masterson
authors: Tao
authors: T
authors: Yu
authors: Lei
authors: L
authors: Shi
authors: Yi
authors: Y
authors: Wang
authors: Martin
authors: M
authors: Gustavsson
authors: Michael M
authors: MM
authors: Mueller
authors: Gianluigi
authors: G
authors: Veglia
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D000099211:Phospholamban