A role for protein phosphorylation in CYP3A4 ubiquitin‐dependent proteasomal degradation (UPD)

Cytochromes P450 (P450s) are known to incur phosphorylation. The precise role of this posttranslational modification is however unclear. Various roles have been ascribed, including marking P450s for degradation. Indeed, we have previously found that CYP3A4, the major human liver P450, and its rat orthologs not only are phosphorylated in the course of their UPD, but also this phosphorylation is enhanced after their structural inactivation. Peptide mapping coupled with mass spectrometric analyses of in vitro phosphorylated CYP3A4 have identified three major target sites: T 264 , S 420 and S 478 . To determine whether phosphorylation of these residues is relevant to in vivo CYP3A4 degradation, we heterologously expressed wild type and CYP3A4 with single, double or triple mutations of these residues to Ala in S. cerevisiae pep4 Δ‐strains with a functional UPD but nonfunctional vacuolar degradation, after verifying that such mutations did not affect CYP3A4 structure and/or function. Our findings revealed that relative to CYP3A4, its S 478 A mutant was significantly stabilized in these yeast, and this stability was greatly to markedly enhanced for its S 478 A/T 264 A, S 478 A/S 420 A and S 478 A/T 264 A/S 420 A double and triple mutants. Together these findings indicate that phosphorylation of a buried, otherwise concealed SRS‐6 S 478 residue by cytosolic kinases is important for CYP3A4 UPD. Supported by NIH GM44037.