Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Vitronectin is a key plasma protein involved in cell adhesion and migration.
• Previous studies indicated Ser378 in vitronectin's heparin-binding domain is phosphorylated by plasma kinases.
• The functional significance of vitronectin phosphorylation remained largely unexplored.

Purpose of the Study:

• To investigate the phosphorylation status of vitronectin during its biosynthesis.
• To identify the specific kinase responsible for Ser378 phosphorylation in vitro.
• To compare the extent of in vitro phosphorylation with that observed in plasma.

Main Methods:

• Analysis of phosphate content in purified plasma vitronectin.
• In vitro phosphorylation assays using purified vitronectin and cAMP-dependent protein kinase.
• Investigation of phosphorylation in HepG2 cells during vitronectin biosynthesis.
• Assessment of heparin's effect on Ser378 phosphorylation at neutral pH.

Main Results:

• Purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein.
• Vitronectin undergoes phosphorylation during biosynthesis in HepG2 cells.
• cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain vitronectin.
• Heparin did not influence the phosphorylation of Ser378 at neutral pH.
• In vitro phosphorylation by cAMP-dependent protein kinase exceeded levels found in plasma.

Conclusions:

• Vitronectin is phosphorylated during its biosynthesis.
• Ser378 is a specific site for phosphorylation by cAMP-dependent protein kinase in single-chain vitronectin.
• In vitro phosphorylation provides a model to study the functional consequences of vitronectin phosphorylation.