Identification and Characterization of an E2 Ubiquitin-Conjugating Enzyme in GCM1 Ubiquitination
Date Issued
2008
Date
2008
Author(s)
Chiang, Meng-Hsiu
Abstract
Protein ubiquitination involves E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ligase. GCM1, whose activity has been demonstrated to be regulated by the ubiquitin-proteasome system, is an essential transcription factor in placental development. Our previous studies have shown that GCM1 is a substrate for the SCFFBXW2 E3 complex, which specifically recognizes GCM1 via the human F-box protein, FBW2. However, the ubiquitin-conjugating enzyme (E2) involved in GCM1 ubiquitination remains elusive, in this study, we first showed that the HeLa S-100 extract can promote ubiquitination of GCM1 in vitro, and then identified an E2 (termed E2-6) specifically regulates GCM1 ubiquitination after screening a panel of E2s. In vitro ubiquitination assay of GCM1 also demonstrated that E1, E2-6, and SCFFBW2 are necessary for GCM1 ubiquitination. Mutation on the catalytic site of E2-6 (E2-6CA) resulted in loss of its interaction with SCFFBW2 and the ubiquitination of GCM1. Moreover, small hairpin RNA-mediated knockdown of E2-6 reduced ubiquitination of GCM1 and subsequently increased GCM1 protein stability in 293T cells. These results reveal that E2-6 is crucial for ubiquitination of GCM1 in vitro and in vivo.
Subjects
Placenta, GCM1, SCF complex, FBXW2, E2-6
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