CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum (ER)-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 ER-associated degradation (ERAD) via the ubiquitin (Ub)-dependent 26S 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... More
CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum (ER)-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 ER-associated degradation (ERAD) via the ubiquitin (Ub)-dependent 26S 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 (pS/pT) by PKA and/or PKC accelerates/enhances its Lys (K)-ubiquitination by either of these E2/E3 systems. Intriguingly, CYP3A4 pS/pT and ubiquitinated K-residues reside within cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu (D/E) clusters, leading us to propose that such post-translational S/T 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 D/E/pS/pT surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2/E3 subcomponents. By imparting additional negative charge to these D/E clusters, such S/T phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2/E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and ERAD. While 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.Copyright 2014, The American Society for Biochemistry and Molecular Biology.
