Calcium-regulated phosphorylation in synaptosomal cytosol: dependence on calmodulin

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Synaptosomal cytosolic proteins undergo phosphorylation, a key post-translational modification.
• Calcium ions play a critical role in regulating various cellular processes, including neuronal signaling.

Purpose of the Study:

• To investigate the role of calcium in stimulating the phosphorylation of specific synaptosomal cytosolic proteins.
• To identify the proteins involved and the regulatory mechanisms of calcium-stimulated phosphorylation.

Main Methods:

• Utilized exogenous calcium and a calcium chelator to assess calcium's effects on protein phosphorylation.
• Employed affinity chromatography with fluphenazine-Sepharose to isolate and study calmodulin's role.
• Measured phosphate incorporation into proteins using various molecular weights.

Main Results:

• Calcium (1.0-100 microM) enhanced protein phosphorylation up to 23-fold, particularly for proteins of 50,000, 55,000, and 60,000 molecular weights.
• Fluphenazine, an antipsychotic, inhibited calcium-stimulated phosphorylation in a concentration-dependent manner.
• Removal of calmodulin abolished calcium-stimulated phosphorylation, which was restored upon calmodulin re-addition.

Conclusions:

• Calcium-dependent protein kinases are vital regulators of specific cytosolic protein phosphorylation in neuronal tissue.
• Calmodulin is essential for mediating calcium-stimulated protein phosphorylation in synaptosomes.
• Identified three key substrate proteins, with one being synaptosome-specific and the others found in both cytosolic and membrane fractions.