Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

Pyerin, Walter; Taniguchi, Hiroyuki

doi:10.1002/j.1460-2075.1989.tb08450.x

Cited by 48 publications

(27 citation statements)

References 49 publications

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“…43 Similar results were obtained for the rat ortholog CYP 2B1, 44 which is the enzyme investigated in the present study. Figure 3 shows the time course of the phosphorylation-dependent inactivation of CYP 2B1.…”

Section: Stimulation Of Hepatocytes By Db-camp Decreases Cyp2b1 Monoosupporting

confidence: 87%

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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Section: Stimulation Of Hepatocytes By Db-camp Decreases Cyp2b1 Monoosupporting

confidence: 87%

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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“…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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“…Using LC-MS/MS analyses, we have identified several CYP2E1 residues, in addition to the previously identified Ser 129 (28,(51)(52)(53) specifically phosphorylated by PKA or PKC in the native enzyme. Furthermore, we document that this phosphorylation is further enhanced after CYP2E1 structural inactivation by cumene hydroperoxide (CuOOH).…”

mentioning

confidence: 81%

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

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Human liver CYP2E1 is a monotopic, endoplasmic reticulumanchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanismbased inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.Hepatic cytochromes P450 (P450s) 2 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. These substrates can modulate P450 content, diversity, and/or function (see Refs. 1, 2 and references therein) through induction via either increased synthesis or protein stabilization, i.e. half-life prolongation (3-9). By contrast, "suicide" substrate/inactivators accelerate the degradation of certain P450s and dramatically curtail their halflives (10 -23). Such substrate-mediated P450 induction and/or enhanced turnover can influence the severity and the time course of certain pharmacokinetic/pharmacodynamic drugdrug interactions and is an important therapeutic consideration (24 -27).P450 turnover has been proposed to involve various proteolytic mechanisms (6 -9, 28 -38). However, it is now increasingly evident that in common with other type I monotopic ER proteins, P450s such as CYPs 3A (both native and structurally inactivated) undergo ER-associated degradation (ERAD) involving the ubiquitin (Ub)-dependent 26 S proteasomal system (UPS) (6 ...

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Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

Cited by 48 publications

References 49 publications

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

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

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

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

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