Identification of HHR23A as a Substrate for E6-associated Protein-mediated Ubiquitination

Abstract

The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in nonsynchronized cells, its levels are regulated in a cell cycledependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.Protein ubiquitination is implicated in a variety of cellular processes, including DNA repair, cell cycle control, chromosomal organization, intracellular translocation of proteins, and apoptosis (1-3). Ubiquitin-dependent proteolysis is the best known aspect of the ubiquitin pathway. The covalent conjugation of multiple ubiquitin molecules to lysine residues of a target protein serves to signal its recognition and rapid degradation by the 26 S proteasome (3-5). Ubiquitination of protein substrates is a multi-step process that involves the concerted action of at least three classes of enzymes as follows: ubiquitinactivating enzyme (E1), 1 ubiquitin-conjugating enzymes (E2s), and ubiquitin protein ligases (E3s) (3). Although the biochemical mechanisms of ubiquitin transfer within the enzymatic components of the pathway and its subsequent conjugation to target proteins is now understood in considerable detail, it is still unclear how specific proteins are recognized by the ubiquitin system as substrates. E1 first activates ubiquitin in an ATP-dependent reaction through the formation of ubiquitin adenylate, followed by a thiol ester bond between the carboxyl terminus of ubiquitin and thiol group of a specific cysteine residue in E1. Ubiquitin is then transferred to a specific cysteine residue in one of several E2s (6). E2 enzymes, in turn, may transfer the ubiquitin either directly to a substrate or to E3 enzymes that finally catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the ⑀-amino group of lysine residues on a target protein (3,7,8). A substrate may be multiply ubiquitinated by sequential linkag...

Human papillomavirus type 16 E6 protein interacts with cystic fibrosis transmembrane regulator-associated ligand and promotes E6-associated protein-mediated ubiquitination and proteasomal degradation

“…It is also strange that CAL was not affected by siE6AP, which was shown to effect hScrib and p53 activation. The level of endogenous HHR23A, an E6-independent target of E6AP was also examined (Kumar et al, 1999), but prominent changes were not detected (data not shown). The inefficiency of siE6AP might be because the silencing effect of siE6AP was not sufficient to affect the steady-state levels of CAL and HHR23A.…”

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

“…As E6AP is involved in the ubiquitination of proteins associated with key cellular functions, [3][4][5][6][7] to examine the biological implications of its suppression in HPV-positive cells, we observed their individual DNA content by FACS analysis. Under normal growth conditions, there was no observable change in the cell-cycle profile associated with ribozyme-mediated reduction in E6AP.…”

“…2 It has since been shown that E6AP acts as an E3 in the ubiquitin-dependent proteolysis of various other cellular proteins, both in the presence and absence of HPV E6. [3][4][5][6][7] E6AP was also the founding member of a family of structurally related E3s carrying a carboxyl terminus that is highly conserved among various organisms, termed the hect domain (homology to E6AP C terminus). 8 Besides its protein ligase function, E6AP…”

“…Recent works from several groups suggest that E6AP may be involved in the regulation of important cellular processes such as transcription, signal transduction, cell survival and/or apoptosis, cell-cycle control and DNA repair. [3][4][5][6][7]9 Hammerhead ribozymes are catalytic RNA molecules that possess conserved catalytic core sequences flanked by two hybridizing arms (stems I and III). 12 Substrate cleavage occurs 3 0 to the nucleotide sequence NUH, where N is any nucleotide and H can be any nucleotide except G. The high degree of specificity and efficiency in substrate binding and cleavage makes ribozymes a valuable tool for gene function analysis.…”

Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex

“…In a screen of both fetal brain and HeLa cell cDNA libraries, several known E6/ E6-AP interactors were identified, including p53, HHR23A (human homolog of rad23), and a homolog of the E6TP1 gene (Unigene Hs. 406879; Scheffner et al 1993;Gao et al 1999;Kumar et al 1999). In addition, a new E6/E6-AP target protein identified in the screen was NFX1 (nuclear factor binds to the X1 box), a transcriptional repressor of MHC class II genes (Song et al 1994).…”