Phosphorylation of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase protein kinase C and cAMP dependent protein kinase

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of ³²PO₄ was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of ³²PO₄/ mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of ³²PO₄/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M_r of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M_r of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.