Figure 3: CRL4^DCAF1 binds to and poly-ubiquinates PP2A-A for degradation.

(a) Inhibition of PP2A activity by okadaic acid rescued GVBD defects of both Ddb1^oo−/− and Dcaf1^oo−/− oocytes. Total numbers of oocytes used (n) are indicated. Error bars indicate s.e.m. *P<0.05, ***P<0.001, Student’s t-test (b,c). Western blotting results showing PP2A-A accumulation, but not PP2A-B and -C subunits, in Ddb1^oo−/− (b) or Dcaf1^oo−/− (c) oocytes at the GV stage. ERK1/2 was used as a protein loading control. (d) RNAi depletion of Dcaf1, Ddb1 and Cul4a/b in HeLa cells resulted in increased PP2A-A protein levels. Samples were collected at 24 h after siRNA transfection. (e) RNAi depletion of Dcaf1 and Ddb1 in HeLa cells blocked PP2A-A degradation after cyclohexamide (100 μg ml⁻¹) inhibition. Samples were collected at 0, 6, 12 and 24 h after cyclohexamide treatment. (f) Co-IP results showing the interaction between DCAF1 and PP2A-A isoforms. HeLa cells were co-transfected with MYC-DCAF1 and FLAG–PPP2R1A/B expression plasmids for 48 h. Target proteins were immunoprecipitated using anti-FLAG agarose beads and subjected to western blotting. Input cell lysates (10%) were immunoblotted with an anti-MYC antibody to show equivalent expression of DCAF1 in all samples. (g) PP2A-A poly-ubiquitination levels were increased after overexpressing CRL4^DCAF1 components. HeLa cells were co-transfected with FLAG–PPP2R1A/B, HA-Ub, and the indicated MYC-tagged protein expression plasmids for 48 h. Target proteins were immunoprecipitated using anti-FLAG agarose beads and subjected to western blotting. (h) RNAi depletion of Dcaf1/Ddb1 resulted in reduced PP2A-A poly-ubiquitination. (i) In vitro binding assay showing that PP2A-A directly interacts with the N terminus of DCAF1. HeLa cells were transfected with FLAG–PPP2R1A expression plasmids for 48 h. Target proteins were immunoprecipitated using anti-FLAG agarose beads. His-tagged N or C terminus of DCAF1 was expressed and purified from E. coli cells. n.s., a non-specific band blotted using an anti-His6 antibody, used as a loading control. (j) PP2A-A is directly poly-ubiquitinated by CRL4^DCAF1 in vitro. HeLa cells were transfected with FLAG–PPP2R1A or FLAG-DCAF1 expression plasmids for 48 h. Target proteins were immunoprecipitated using anti-FLAG agarose beads separately and subjected to ubiquitination assay in the presence of E1, E2 and Ubiquitin.