Phosphorylation of cytochrome P450: regulation by cytochrome b5.
Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.
Epstein
P M
PM
Department of Pharmacology, University of Connecticut Health Center, Farmington 06032.
Curti
M
M
Jansson
I
I
Huang
C K
CK
Schenkman
J B
JB
D000818:Animals; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000494:pharmacology*; D015786:Cytochromes b5; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D065607:Cytochrome P-450 Enzyme Inhibitors; D003573:Cytochrome b Group; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C