FIG 6.

COP1 E3 ligase is involved in VRK2-mediated TRiC protein turnover. (A) Immunoprecipitation assay using HEK293T cells cotransfected with Flag-COP1 and HA-CCT4 vectors. Encoded HA-CCT4 was coimmunoprecipitated with an anti-Flag antibody. (B) VRK2-mediated downregulation of TRiC disappeared in cells transfected with siCOP1. (C) GST pulldown assay using in vitro-purified SUMO-VRK2 and GST-RBX1 recombinant proteins. (D) VRK2 enhanced CCT4 ubiquitination by the COP1 E3 ligase supercomplex in vitro. Purified HIS-CCT4 was incubated with components of the ubiquitin system in the presence or absence of the COP1 E3 ligase supercomplex with or without VRK2. Polyubiquitinated CCT4 was immunoblotted with an anti-Flag antibody to detect Flag-ubiquitin. (E) Knockdown of both VRK2 and COP1 enhanced CCT4 protein stability. Cells were harvested at the indicated times after cycloheximide (40 μg/ml) treatment, and then endogenous CCT4 was detected by Western blotting.