Ca2+, calmodulin-dependent phosphorylation of glycogen synthase by a brain protein kinase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Glycogen synthase is a key enzyme in glycogen synthesis.
• Protein kinases play crucial roles in regulating metabolic enzymes.
• Calcium-calmodulin dependent protein kinases are important signaling molecules in the brain.

Purpose of the Study:

• To investigate the effect of a brain Ca2+, calmodulin-dependent protein kinase on skeletal muscle glycogen synthase.
• To identify the phosphorylation sites and stoichiometry on glycogen synthase.
• To determine the regulatory properties of this kinase-substrate interaction.

Main Methods:

• Purification of Ca2+, calmodulin-dependent protein kinase from brain.
• Enzymatic assay of glycogen synthase phosphorylation.
• Inhibition studies using EGTA and trifluoperazine.
• Cyanogen bromide peptide mapping of phosphorylated glycogen synthase.

Main Results:

• A brain Ca2+, calmodulin-dependent protein kinase (Mr 640,000) phosphorylated skeletal muscle glycogen synthase.
• Phosphate incorporation was 1.4 mol/mol subunit.
• Phosphorylation occurred at serine residues on cyanogen bromide peptides CB-1 and CB-2.
• The reaction was inhibited by EGTA and trifluoperazine, indicating Ca2+ and calmodulin dependence.

Conclusions:

• Brain Ca2+, calmodulin-dependent protein kinase can directly regulate skeletal muscle glycogen synthase.
• Specific serine residues are the targets for phosphorylation.
• The phosphorylation is tightly regulated by calcium and calmodulin levels.