Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes

Bartłomowicz, B; Waxman, David J.; Utesch, Dietmar; Oesch, Franz; Friedberg, Thomas

doi:10.1093/carcin/10.1.225

Cited by 33 publications

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“…The isozyme-specific substrate-dependent protection from phosphorylation and degradation of P450IIE1 in the hepatocytes suggests the mechanism to be of importance for regulation of the specific P450 content in the cell. Definitive proof for a physiological role has to await experiments performed in vivo, although the stabilization mechanism for IIE1 has been reported (6) Previous investigations revealed P4501IB1 to be a target for cAMP-dependent kinase in hepatocytes (18)(19)(20), and phosphorylation of P4501IB1 or IIB4 on Ser-128 (19,21) has been connected to conversion of the protein to the inactive P420 form (22) and to decreased enzymic activity (20). The results herein reported indicate that such phosphorylation is regulated in an isozyme-specific manner by the substrate and might trigger denaturation and heme loss with subsequent apoprotein sorting and degradation.…”

Section: Resultsmentioning

confidence: 99%

Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

Eliasson

Johansson

Ingelman‐Sundberg

1990

Proc. Natl. Acad. Sci. U.S.A.

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The hepatic cytochrome P450 system, with numerous different P450 enzymes, is characterized by its inducibility by a variety of endogenous and exogenous com-pounds. Specific forms ofP450, exhibiting distinct but partially overlapping substrate specificities, are increased in response to a given chemical. Consequently, the rate of elimination of the inducing compound is often enhanced and the system is in this respect adaptive to changes in the environment. Transcriptional activation mechanisms for the endo-or xenobiotically controlled P450 synthesis are well documented. Here we describe a mechanism for posttranslational ligand-dependent stabilization of ethanol-inducible P4501UE1 in hepatocyte cultures. Glucagon or 8-bromoadenosine 3',5'-cyclic monophosphate causes an enhanced rate of P4501E1 degradation in the hepatocytes as well as phosphorylation on Ser-129, a reaction which denatures the protein under in vitro conditions. Substrates for the enzyme, such as ethanol and imidazole, protect the enzyme from phosphorylation and degradation in hepatocytes but do not influence phosphorylation or degradation of phenobarbital-inducible P450IIB1. Our proposed mechanism, which remains to be shown under in vivo conditions, describes the P450 molecules as receptors for the compounds in question and might provide a way by which endo-and xenobiotics regulate their own rate of metabolism.The cytochrome P450 system has the capability to metabolize various endogenous and exogenous substances. In mammals, the P450 gene superfamily comprises at least nine families, with 19 subfamilies in total (1, 2). The various P450 forms are characterized by their specific, but overlapping, substrate specificities and by their inducibility. Transcriptional activation mechanisms for various P450 genes have been described and transcriptional factors regulating expression of P450 molecules belonging to the IA and IIB gene families have been identified (3,4). However, the specific P450 increase in response to elevated levels of a given chemical can also be achieved by posttranslational stabilization (5-7).We have described (7) the posttranslational stabilization of ethanol-inducible P450IIE1 in cultured hepatocytes by isozyme-specific substrates at an efficiency corresponding to the binding affinities of the substrates to the enzyme. The substrate-dependent posttranslational stabilization of P450IIE1 has also been shown to occur in vivo (6). These findings raised the question about the origin of the endogenous systems regulating P450IIE1 degradation. We here present a mechanism by which specific P450IIE1 ligands protect the enzyme from cAMP-dependent phosphorylation and associated denaturation and degradation but do not affect the regulation of phenobarbital-inducible P450IIB1. The results indicate that the mechanism is ofgeneral importance for ligand-dependent control of P450 isozyme levels. (7) and suspended in 50 mM potassium phosphate (pH 7.4) containing 100 mM NaF, 100 mM KCl, 0.2% Nonidet P-40, and 1 mM EDTA (10). Anti-P450IIE1 IgG ...

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Section: Resultsmentioning

confidence: 99%

Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

Eliasson

Johansson

Ingelman‐Sundberg

1990

Proc. Natl. Acad. Sci. U.S.A.

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“…8). Although untreated rat hepatocytes possess low but significant levels of CYP2B1, 28,49,50 in the constitutive situation CYPs other than CYP2B1 or non-CYP enzymes may contribute substantially to the observed effects, potentially further extending the scope of the principle shown in this study. In this context it is interesting that e.g., prostaglandin H synthase (which has recently been found to be expressed in hepatocytes, 51 has been shown to be able to activate CPA.…”

Section: Discussionmentioning

confidence: 73%

“…27 We and others have demonstrated that the major PB inducible CYP2B1 and 2B2 are phosphorylated in intact cells and that this phosphorylation is cAMP-dependent. 28,29 The phosphorylation of CYP2B1 resulted in the downregulation of its activity, 30 showing a novel posttranslational control mechanism for CYP2B1. We have recently shown that cAMP-dependent phosphorylation of CYP2E1 (ethanol-inducible CYP, responsible for activation of many environmental procarcinogens), when tethered to the wildtype phosphoacceptor Ser129, resulted in the reduction of its activity and stabilization of the enzyme protein, whereas substitution of Ser129 by nonphosphorylatable amino acids modified CYP2E1 substrate specificity.…”

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confidence: 99%

cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal controlin vitro andin vivo

et al. 2001

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An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status. © 2001 Wiley-Liss, Inc. Key words: cyclophosphamide; CYP2B1; phosphorylation; hormonal controlThe activity of many of the foreign compound metabolizing enzymes, e.g., CYP, is of obvious importance for both physiologic homeostasis as well as effects of drugs and environmental chemicals. 1 Much of the detoxification is performed by CYP enzymes. Many of them are inducible and have a broad and overlapping substrate specificity. 2 Interindividual differences in the expression level of CYP enzymes are known, 3 thus contributing to the variations in toxic symptoms and therapeutic response evoked by antineoplastic drugs. 4 CYP2B1 (the major phenobarbital-inducible form) has long been suggested as 1 of the most important enzymes involved in the activation of widely used antineoplastic prodrugs. It catalyzes oxidative desulfuration of thio-TEPA to TEPA as well as 1-electron reduction of Adriamycin to its semiquinone free radical [5][6][7] and, most importantly, is responsible for conversion of the oxazaphosphorines CPA and IFA to their active metabolites. 8,9 It has been shown that 3 specific rat liver CYP enzymes (CYP2B1, CYP2C6 [constitutively expressed, gender independent] and CYP2C11 [constitutively expressed, male specific]) are major catalysts of CPA activation in adult rat liver, 8,10 while a rat CYP3A enzyme additionally contributes to the activation of IFA. 9 The corresponding human enzymes CYP2B6 and CYP3A4 activate CPA and IFA in human liver, respectively. 11-13 It was demonstrated that among rat enzymes CYP2B1 (the ortholog of the human CYP2B6) is by far the most active catalyst. 8,11,12 Activation of CPA to the therapeutic active species has its initial step in the liver. 8 The CYP2B1-catalyzed 4-hydroxylation reaction forms a highly unstable metabolite, which undergoes spontaneous ␤-elimination and gives rise to 2 pro...

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“…In the absence of stimulation, the incorporation of radioactive phosphate into cytochromes P450 (isolated and purified after the incubation.with 32P-orthophosphate) was very low. After stimulation by extracellular glucagon or by membrane permeating cAMP-derivatives (N6,02'-dibutyrylcAMP and 8-thiomethyl cAMP) a marked incorporation of 32P-phosphate into cytochrome P450 took place (15). Autoradiography of gel electrophoretically separated proteins from solubilized microsomes of these hepatocytes and of purified cytochromes P450 combined with visualization on Western blots by specific antibodies showed that four cytochromes P450 were phosphorylated, cytochromes P450 2B1 and2B2, and two cytochromes P450 2B1-related proteins, one of them inducible by phenobarbital (15).…”

Section: Introductionmentioning

confidence: 99%

“…After stimulation by extracellular glucagon or by membrane permeating cAMP-derivatives (N6,02'-dibutyrylcAMP and 8-thiomethyl cAMP) a marked incorporation of 32P-phosphate into cytochrome P450 took place (15). Autoradiography of gel electrophoretically separated proteins from solubilized microsomes of these hepatocytes and of purified cytochromes P450 combined with visualization on Western blots by specific antibodies showed that four cytochromes P450 were phosphorylated, cytochromes P450 2B1 and2B2, and two cytochromes P450 2B1-related proteins, one of them inducible by phenobarbital (15). Pyerin and Taniguchi (16) and Koch and Waxman (17) have also observed that the major phenobarbital-inducible cytochrome P450 is phosphorylated in the intact hepatocytes and in the whole animal (17) where phosphorylation took place in the liver in the absence of any exogenous stimulation and was increased by N6, 02' -dibutyryl-cAMP and theophyllin.…”

Section: Introductionmentioning

confidence: 99%

Mechanism-based predictions of interactions.

Oesch

Oesch-Bartlomowicz

Arens

et al. 1994

Environ Health Perspect

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Exposure to more than one toxic compound is common in real life. The resulting toxic effects are often more than the simple sum of the effects of the individual compounds. It is unlikely that it will ever be possible to test all combinations. It is therefore highly desirable to improve or develop means for reasonably approximating predictions of interactions. In order to be valid and extrapolatable, these predictions are most promising if they are mechanism-based. Examples will be given for possibilities of mechanism-based predictions of interactions which exceed trivialities of simple increases by enzyme induction of enzymatic rates of a given biotransformation pathway leading to a toxic metabolite. Instead, examples will be provided where competition between various enzymes for shunting the same substrate into divergent pathways can lead to predictable dramatic changes in toxicity by shifting the metabolic routes under conditions of no significant changes of overall metabolism. Further examples are given on predictable interactions between chemicals which need bioactivation for exerting their toxicity and chemicals which effect hormonal status and other endogenous factors which in turn modify enzymes involved in the control of toxic metabolites.-Environ Health Perspect 102 (Suppl 9): 5-9 (1994)

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Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes

Cited by 33 publications

References 0 publications

Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal controlin vitro andin vivo

Mechanism-based predictions of interactions.

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