A role for protein phosphorylation in cytochrome P450 3A4 ubiquitin-dependent proteasomal degradation.
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.
Wang
Yongqiang
Y
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA.
Liao
Mingxiang
M
Hoe
Nicholas
N
Acharya
Poulomi
P
Deng
Changhui
C
Krutchinsky
Andrew N
AN
Correia
Maria Almira
MA
D000818:Animals; D002846:Chromatography, Affinity; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D016296:Mutagenesis; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013104:Spheroplasts / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D018121:Receptors, Cytokine; D025801:Ubiquitin; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C555000:AMFR protein, human; C555002:Amfr protein, mouse; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex