Figure 3. Cyclin E (cyc E) is conjugated to SUMO2/3 on chromatin independently of Cdk2 kinase activity.

(a) Cyc E-immunodepleted S-phase Xenopus extracts (ΔE; ND, mock depletion) were supplemented with [³⁵S]cyc E (translated in Cdk2-depleted egg extracts) and associated with recombinant Cdk2 (ΔE+E/K2). Cyc E levels were analysed by western blotting using anti-cyc E antibodies, and quantified by phosphorimaging (left panels). Radioactive cyc E was immunoprecipitated from chromatin isolated from 400 μl of replication reactions supplemented with 5 μM SUMO1-VS. Immunoprecipitates were split in two and treated or not with 1 μg GST-SENP (30 °C, 20 min). [³⁵S]cyc E and SUMOylated proteins were detected on the same blot by phosphorimaging and using anti-SUMO2/3 antibodies, respectively (right panels). (b) Replicating chromatin was prepared from 50 μl replication assays (30-min time-point) carried out in the presence of 5 μM SUMO1-VS or 250 ng μl⁻¹ Ubc9dn and 60 ng μl⁻¹ GST-SENP as indicated. Endogenous cyc E and SUMO2/3 conjugates were detected by western blotting. (c) RNF4 pull-downs (using beads coupled to wild-type (wt) or mutant (mut) RNF4) were performed as described in Methods and analysed by western blotting (d) Aliquots of [³⁵S]cyc E (5 μl) translated in Cdk2-depleted egg extracts (−) were supplemented with 5 μM SUMO1-VS and with or without a SUMOylation mix. After 30 min, GST-Cdk2 was added in one reaction (+) for 15 min. [³⁵S]cyc E was detected by phosphorimaging. (e) [³⁵S]cyc E translated and detected as in d was mixed with GST-Cdk2 (wt) or with the kinase-dead mutant GST-Cdk2 K33R (KD), as indicated. Samples of both complexes were reconstituted in egg extracts and incubated with (+) or without (−) a SUMOylation mix (left panel). Lambda phosphatase (λP) was added or not for 30 min after the SUMOylation reaction (right panel). (f) Replicating chromatin was isolated from 30-min replication assays supplemented with 5 μM SUMO1-VS and with or without 100 μM Nu6102, a specific Cdk2 inhibitor. Cyc E and SUMO2/3 conjugates were detected by western blotting. The asterisk indicates the mobility shift of the predominant polySUMOylated cyc E form. (g) Replicating chromatin was resuspended in CPB/1% SDS, 10-fold diluted in CPB/1% Triton X-100 and then used for immunoprecipitation (IP) experiments using anti-cyc E antibodies (E) or non-immune rabbit IgG (c).