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. Author manuscript; available in PMC: 2014 Dec 23.
Published in final edited form as: Nat Commun. 2014 Jun 23;5:4234. doi: 10.1038/ncomms5234

Figure 8. CDK1 activity phosphorylates UBC9 for CDK6 sumoylation.

Figure 8

(a) YFP-SUMO1 and Flag-CDK6 expressing LN229 cells were immunoprecipitated in the hours after released from nocodazole synchronization to the cell cycle as monitored by flow cytometry (low panel). Flag immunoprecipitates (IP) and whole cell lysate (WCL) were analyzed by western blotting with asynchronized (AS) cells used as controls (top panel). (b) LN229 cells were co-transfected with YFP-SUMO1 and Flag-CDK6, treated with nocodazole for 20 hrs and then treated or not with RO-3306 for the indicated hrs (top). Flag IP and WCL were analyzed by western blotting using the indicated antibodies (left). (c) In vitro sumoylation of CDK6 in the absence and presence of the cyclin B1-CDK1 complex (CDK1/Cycl B1). (d) HEK293 cells were expressed with the tagged proteins, immunoprecipitated and analyzed by western blotting (top) and CDK1 kinase assay (bottom). (e) Flag-CDK6 was transfected with Myc-UBC9 wt or S71D mt in LN229 cells, immunoprecipitated using Flag antibodies and tested by western blotting for UBC9-CDK6 interaction. (f) LN229 cells were transfected with Myc-UBC9 WT and S71A mt together with Flag-CDK6 and/or YFP-SUMO1, immunoprecipitated and examined by western blotting. (g) The molecular model shows that CDK1 phosphorylates UBC9 and thus enhances SUMO1-CDK6 conjugation from G2/M to G1 phase, driving the cell cycle through G1/S transition.