Phosphorylation of cytochrome-P-450-dependent monooxygenase components.

Most chemical carcinogens require activation by polysubstrate monooxygenase. The phosphorylation of essential components of this cytochrome P-450 monooxygenase system, isolated from rabbit liver microsomes, cytochrome P-450 (LM2) and cytochrome reductase, was tested using two different protein kinases. One of the kinases, a cyclic AMP-independent phosvitin kinase (kinase P), was inactive in all systems tested. However, the catalytic subunit of a cyclic AMP-dependent protein kinase (kinase C) catalyzed phosphoryl group transfer to both proteins, but to different extents. Cytochrome P-450 was phosphorylated when added as sole component and also when in the presence of P-450 reductase and phosphatidylcholine. In contrast, the weak phosphorylation of P-450 reductase was reduced considerably in a complete reconstituted system containing P-450 and phosphatidylcholine. The inclusion of kinase P did not alter these results which excludes the possibility that these kinases participate in a sequential phosphorylation mechanism. The monooxygenase constituents themselves were without kinase activity. When hepatic microsomes were isolated in presence of the phosphatase inhibitor sodium fluoride no significant change in monooxygenase (7-ethoxycoumarin O-deethylation) activity was observed, whilst after preincubation with either acid or alkaline phosphatase a significant reduction in monooxygenase activity was measured. Thus, cytochrome P-450 (LM2) is phosphorylatable by protein kinase C and the catalytic activity of polysubstrate monooxygenase decreases after preincubation of microsomes with phosphatases.