Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes

• Published in: The Journal of biological chemistry Vol. 290; no. 6; pp. 3308 - 3332
• Main Authors: Wang, YongQiang, Kim, Sung-Mi, Trnka, Michael J, Liu, Yi, Burlingame, A L, Correia, Maria Almira
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 06.02.2015
• Subjects: Amino Acid Sequence; Animals; Cytochrome P-450 CYP3A - chemistry; Cytochrome P-450 CYP3A - genetics; Cytochrome P-450 CYP3A - metabolism; HEK293 Cells; Humans; Mice; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Protein Synthesis and Degradation; Receptors, Autocrine Motility Factor - metabolism; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; E3 Ubiquitin Ligase; Ubiquitin-conjugating Enzyme (E2 enzyme); CHIP; Cytochrome P450; Mass Spectrometry (MS); Endoplasmic Reticulum-associated Protein Degradation (ERAD); gp78/AMFR; Protein Phosphorylation; CYP3A4; Ubiquitylation (ubiquitination)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.611525

CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation.