A Role for Protein Phosphorylation in Cytochrome P450 3A4 Ubiquitin-dependent Proteasomal Degradation

Yongqiang Wang,Mingxiang Liao,Nicholas Hoe,Poulomi Acharya,Changhui Deng,Andrew N Krutchinsky,Maria Almira Correia

doi:10.1074/jbc.m806104200

Yongqiang Wang, Mingxiang Liao + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m806104200

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Abstract

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr(264) and Ser(420). We now document that liver cytosolic kinases additionally target Ser(478) as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Delta strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser(478), Thr(264), and Ser(420) residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.

Highlights

Hepatic cytochromes P450 (P450s)3 are integral endoplasmic reticulum (ER)-anchored hemoproteins engaged in the oxidative biotransformation of various endo- and xenobiotics
On the basis of rapid phosphorylation of CuOOH-inactivated CYP3A4 that precedes its ubiquitination and 26 S proteasomal degradation in an in vitro liver cytosolic fraction II (FII)-catalyzed system, we have proposed that CYP3A4 phosphorylation was essential for targeting it to proteins participating in its UPD/ER-associated degradation (ERAD) [13]
Rat Liver Cytosolic FII-mediated CYP3A4 Phosphorylation, Mass Spectrometric Analyses—Following incubation with rat liver cytosolic FII, CYP3A4 protein isolated with TALON Dynabeads was subjected to mapping of phosphorylation sites by mass spectrometry

Summary

The abbreviations used are

P450, CYPs, cytochrome P450; CuOOH, cumene hydroperoxide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FII, fraction II; HMM, high molecular mass; LC-MS/MS, liquid chromatography-tandem mass spectrometry; Lys-C, lysylendopeptidase C; PKA, cAMP-dependent protein kinase A; PKC, protein kinase C; Ub, ubiquitin; UBC, genes for Ub-conjugating enzymes; UPD, Ub-dependent proteasomal degradation; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; DTT, dithiothreitol. In vitro model studies of CYP3A4 with PKC as the kinase, coupled with lysylendopeptidase C (Lys-C) digestion of the phosphorylated protein and liquid chromatography-tandem mass spectrometric (LC-MS/MS) analyses of the Lys-C digests, identified two PKC-phosphorylated CYP3A4 peptides 258ESRLEDpTQK266 and 414FLPERFpSK421 unambiguously phosphorylated at Thr264 and Ser420 [14] These same residues were phosphorylated in corresponding studies with PKA.. Using an in vitro reconstituted CYP3A4 ubiquitination system, catalyzed by human Ub-conjugating E2 enzyme UBC7 and integral ER protein gp as the E3 Ub ligase [12], we document that PKA/PKC-mediated phosphorylation of the wild type CYP3A4 (CYP3A4wt) considerably enhanced its UBC7/gp78-mediated ubiquitination Together these findings reveal the critical importance of CYP3A4 phosphorylation at these residues for its UPD and suggest a direct link between phosphorylation and its ubiquitination and degradation

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