And-1 prevents the ubiquitination of Gcn5 by CRL4Cdt2 E3 ligase.
A, overexpression of Cdt2 enhances the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested 48 h after transfection. Cells were treated with MG132 for 6 h before harvest. The ubiquitinated species were precipitated by HA antibodies and subjected to immunoblot for ubiquitinated FLAG-Gcn5 using anti-FLAG antibody. Ub, ubiquitin. B, overexpression of And-1 suppresses the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested for 48 h and treated as in A. Note that And-1 overexpression increased the FLAG-Gcn5 protein levels and reduced the ubiquitination of Gcn5. IP, immunoprecipitation; LC, loading control. C, And-1 prevents Gcn5 ubiquitination by CRL4Cdt2
in vitro. Incubation of immunopurified CRL4Cdt2 with Gcn5 in an in vitro ubiquitin ligase assay increased the formation of Gcn5 ubiquitinated species. The addition of immunopurified And-1 suppressed the Gcn5 ubiquitination by CRL4Cdt2. IF, immunofluorescence. D, And-1 overexpression suppresses the ubiquitination of the C terminus of Gcn5. U2OS cells transfected with the indicated plasmids were harvested. FLAG-And-1(1–491) or FLAG-And-1(492–837) was precipitated using anti-FLAG M2-agarose beads. FLAG precipitates and inputs were subjected to immunoblot using anti-ubiquitin, anti-FLAG, or anti-And-1 antibody.