Protein kinase and its endogenous substrates in coated vesicles

Area of Science:

• Biochemistry
• Cell Biology
• Neuroscience

Background:

• Coated vesicles are crucial for intracellular transport.
• Protein phosphorylation plays a key role in regulating cellular processes.

Purpose of the Study:

• To characterize the protein kinase activity associated with bovine brain coated vesicles.
• To identify the substrates and localization of this kinase.

Main Methods:

• Preparation of coated vesicles from bovine brains.
• Phosphorylation assays using endogenous and exogenous protein substrates.
• Subfractionation of coated vesicles into coat and core components.
• Enzyme activity assays under various ionic conditions.

Main Results:

• A protein kinase activity was identified in coated vesicles, phosphorylating Mr 150,000, 120,000, 48,000, and 32,000 proteins.
• The Mr 48,000 protein was the most strongly phosphorylated endogenous substrate.
• The kinase activity was independent of cyclic nucleotides and Ca2+/calmodulin but stimulated by Mg2+.
• Kinase activity was predominantly localized to the outer coat subfraction of coated vesicles.

Conclusions:

• Bovine brain coated vesicles contain a distinct protein kinase activity.
• This kinase primarily targets specific endogenous structural proteins, with a preference for the Mr 48,000 protein.
• The kinase is localized to the coat fraction, suggesting a role in coat assembly or function.