Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).
The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.
Rolfe
M
M
Mitotix Inc., Cambridge, MA 02139, USA.
Beer-Romero
P
P
Glass
S
S
Eckstein
J
J
Berdo
I
I
Theodoras
A
A
Pagano
M
M
Draetta
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D014764:Viral Proteins; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases