Eliasson E, Mkrtchian S, Halpert J R, Ingelman-Sundberg M
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
J Biol Chem. 1994 Jul 15;269(28):18378-83.
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.
已知大鼠主要的糖皮质激素诱导型细胞色素P450（CYP3A1）在转录水平受糖皮质激素调控，在翻译后水平受底物依赖性稳定作用调控。我们使用原代肝细胞、从地塞米松处理的大鼠分离的肝微粒体和纯化的酶，研究了底物/配体稳定作用的机制。用胰高血糖素处理肝细胞导致CYP3A1磷酸化增加3倍，同时酶的降解速率加快。特异性CYP3A1底物或配体，如红霉素、三乙酰竹桃霉素和克霉唑（CTZ）可保护肝细胞中的酶不被降解，并同时抑制（r = 0.99）胰高血糖素诱导的酶磷酸化。用纯化的CYP3A1和分离的肝微粒体进行的体外实验显示，一个主要位点（Ser393）被cAMP依赖性激酶的催化亚基磷酸化，该反应受配体抑制。微粒体实验表明存在一种对CYP3A1有活性的内源性cAMP依赖性激酶。添加外源性cAMP依赖性激酶可增加微粒体CYP3A1的磷酸化速率，该反应进一步受到NADPH的刺激，但受CTZ抑制。分光光度法显示，微粒体磷酸化导致细胞色素P450明显变性，而CTZ可保护其免受此反应影响。监测睾酮的CYP3A1依赖性6β-羟化反应时也观察到类似效果。提示细胞CYP3A1水平在很大程度上在翻译后受Ser393上底物调节的cAMP依赖性磷酸化调控，随后在内质网中变性和降解。
