Subunit structure and multiple phosphorylation sites of phospholamban

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiology

Background:

• Phospholamban regulates cardiac sarcoplasmic reticulum Ca2+-ATPase activity.
• Phosphorylation is a key mechanism for modulating phospholamban function.

Purpose of the Study:

• To investigate the phosphorylation sites and stoichiometry of phospholamban using different protein kinases.
• To elucidate the structural organization of phospholamban monomers and multimers.

Main Methods:

• Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze mobility shifts.
• Pulse-chase experiments with centrifuge column method to track phosphorylation.
• Two-dimensional tryptic peptide mapping to identify phosphorylation sites.

Main Results:

• cAMP-dependent phosphorylation induced five mobility shifts in high molecular weight phospholamban and one in the low molecular weight form.
• Ca2+-calmodulin-dependent phosphorylation induced ten mobility shifts in high molecular weight phospholamban and two in the low molecular weight form.
• Peptide mapping revealed cAMP-dependent kinase phosphorylates site A, while Ca2+-calmodulin-dependent kinase phosphorylates sites C1 and C2.

Conclusions:

• Phosphorylation sites for cAMP-dependent and Ca2+-calmodulin-dependent protein kinases are distinct on the phospholamban molecule.
• The high molecular weight phospholamban is likely a pentamer of low molecular weight monomers, each with specific phosphorylation sites.