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Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes. 

Abstract The E6 protein of the human papillomavirus (HPV) underpins important protein interaction networks between the virus and host to promote viral infection. Through its interaction with E6AP, a host E3 ubiquitin (UB) ligase, E6 stirs the protein ubiquitination pathways toward the oncogenic transformation of the infected cells. For a systematic measurement of E6 reprogramming of the substrate pool of E6AP, we performed a proteomic screen based on “orthogonal UB transfer (OUT)” that allowed us to identify the ubiquitination targets of E6AP dependent on the E6 protein of HPV‐16, a high‐risk viral subtype for the development of cervical cancer. The OUT screen identified more than 200 potential substrates of the E6‐E6AP pair based on the transfer of UB from E6AP to the substrate proteins. Among them, we verified that E6 would induce E6AP‐catalyzed ubiquitination of importin proteins KPNA1‐3, protein phosphatase PGAM5, and arginine methyltransferases CARM1 to trigger their degradation by the proteasome. We further found that E6 could significantly reduce the cellular level of KPNA1 that resulted in the suppression of nuclear transport of phosphorylated STAT1 and the inhibition of interferon‐γ‐induced apoptosis in cervical cancer cells. Overall, our work demonstrates OUT as a powerful proteomic platform to probe the interaction of E6 and host cells through protein ubiquitination and reveals a new role of E6 in down‐regulating nuclear transport proteins to attenuate tumor‐suppressive signaling.