Substrate-regulated, cAMP-dependent phosphorylation, denaturation, and degradation of glucocorticoid-inducible rat liver cytochrome P450 3A1.
The major rat glucocorticoid-inducible cytochrome P450 (CYP3A1) is known to be regulated at a transcriptional level by glucocorticoids and at a post-translational level by substrate-dependent stabilization. We have investigated mechanisms of substrate/ligand stabilization using primary hepatocytes, isolated liver microsomes from dexamethasone-treated rats, and purified enzymes. Treatment of hepatocytes with glucagon caused a 3-fold increase in CYP3A1 phosphorylation as well as an enhanced degradation rate of the enzyme. Specific CYP3A1 substrates or ligands, such as erythromycin, triacetyloleandomycin, and clotrimazole (CTZ) protected the enzyme from degradation in hepatocytes and inhibited in a concomitant manner (r = 0.99) glucagon-induced phosphorylation of the enzyme. In vitro experiments with purified CYP3A1 and isolated liver microsomes revealed one major site (Ser393) phosphorylated by the catalytic subunit of cAMP-dependent kinase, a reaction inhibited by ligands. Experiments in microsomes showed the presence of an endogenous cAMP-dependent kinase active on CYP3A1. Addition of exogenous cAMP-dependent kinase increased the rate of microsomal CYP3A1 phosphorylation, a reaction further stimulated by NADPH, but inhibited by CTZ. The microsomal phosphorylation caused a pronounced denaturation of cytochrome P450, as revealed spectrophotometrically, whereas CTZ protected from this reaction. Similar effects were noted when the CYP3A1-dependent 6 beta-hydroxylation of testosterone was monitored. It is suggested that the cellular CYP3A1 level is regulated to a significant extent posttranslationally by substrate-regulated cAMP-dependent phosphorylation on Ser393, followed by denaturation and degradation in the endoplasmic reticulum.
Eliasson
E
E
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Mkrtchian
S
S
Halpert
J R
JR
Ingelman-Sundberg
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D004790:Enzyme Induction; D005260:Female; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011489:Protein Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D010446:Peptide Fragments; D010748:Phosphopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D013739:Testosterone; D012694:Serine; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; D017868:Cyclic AMP-Dependent Protein Kinases