Figure 3.

CDK1-Cyclin B1 Phosphorylates and Activates RNMT

HeLa cells were transfected with CDK1, CDK3, cyclin B1, and cyclin E1 siRNA.

(A and B) After 48 hr, (A) pT77 and total RNMT levels were analyzed in RNMT immunoprecipitates, and (B) CDK and cyclin levels were analyzed in cell extracts by WB. c, control.

(C) Recombinant His-RNMT (WT or T77A)-GST-RAM was incubated with activated CDK1-cyclin B1 and ³²P-ATP for 60 min and resolved by SDS-PAGE. Labeled bands were visualized by phosphoimaging. pT77 and total RNMT were visualized by WB and Coomassie stain.

(D) To assess enzyme kinetics, the CDK1-cyclin B1 kinase reaction was performed using a titration of RNMT or histone H1 over a time course (Figure S1). The charts depict reaction velocities for phosphorylation of RNMT-RAM or histone H1. Error bars represent SD for reaction velocity at each substrate concentration. k_cat and K_M values were calculated using an allosteric sigmoidal curve fit.

(E) As in (C), except a time course experiment was performed. Quantitation of the ATP:RNMT incorporation ratio is reported above.

(F) His-RNMT-GST-RAM was incubated with activated CDK1-cyclin B1 and ATP for 10 min as in (E) and then utilized in the cap methyltransferase assay. rxn, reaction.

(G) His-RNMT WT or 77A-GST-RAM were incubated in the presence and absence of ATP and CDK1-cyclin B1 for 10 min as above and then utilized in the cap methyltransferase assay.

Average and SD for three independent experiments is depicted. ^∗p < 0.05 ^∗∗p < 0.005, t test. See also Figure S1.