Cholesterol 7alpha-hydroxylase is phosphorylated at multiple amino acids

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Metabolism

Background:

• Cholesterol 7alpha-hydroxylase (gpCYP7A1) is the rate-limiting enzyme in bile acid synthesis.
• Its activity is hypothesized to be modulated by phosphorylation and dephosphorylation events.

Purpose of the Study:

• To investigate the role of phosphorylation in regulating gpCYP7A1 activity.
• To identify specific kinases that phosphorylate gpCYP7A1.
• To elucidate the mechanism linking cholesterol synthesis and degradation.

Main Methods:

• Utilized human hepatoma HepG2 cells to study bile acid production.
• Employed FeIII affinity chromatography to isolate phosphoproteins.
• Performed immunoblotting with radioactive orthophosphate labeling.
• Expressed recombinant gpCYP7A as 6xHIS fusion proteins.
• Conducted in vitro kinase assays using various kinases, including AMPK, JNK, PKA, and PKC isoforms.

Main Results:

• Kinase activators significantly reduced bile acid production in HepG2 cells.
• gpCYP7A1 was identified in the phosphoprotein fraction of HepG2 cell extracts.
• Immunoblots confirmed strong labeling of gpCYP7A1 with radioactive orthophosphate.
• AMP-activated protein kinase (AMPK) directly phosphorylated recombinant gpCYP7A1.
• Phosphorylation sites were mapped using synthetic peptides and kinase screening.

Conclusions:

• Phosphorylation is a key regulatory mechanism for gpCYP7A1 activity.
• AMPK directly phosphorylates gpCYP7A1, establishing a link between cholesterol synthesis and degradation pathways.
• This finding provides a potential mechanism for coordinating cholesterol homeostasis.