Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo.
Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)
Koch
J A
JA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Waxman
D J
DJ
D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010105:Oxygenases / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011916:Rats, Inbred F344
D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D011494:Protein Kinases; D010634:Phenobarbital