Figure 3. S6K1 phosphorylates CDK1 and MSH6 in vitro.

(a) In vitro kinase assay using γ-³²P-ATP and recombinant His-S6K1 [T412E] with either recombinant His-Cdk1 (CDK1), or recombinant His-MSH6 (MSH6, 1-718aa fragment as described in Materials and methods) in the indicated combinations. First three panels show reaction products resolved by SDS-PAGE and visualized (the second and third panels are short and long exposures of the same membrane). The fourth panel shows the same membrane as the first panel probed with anti-Cdk1 antibody and detected by enhanced chemiluminescence. Nonspecific radioactive band is marked by <. (b) Non-radioactive in vitro kinase assay using recombinant His-S6K1 [T412E] with either recombinant GST-S6 (S6), His-Cdk1(Cdk1) or His-MSH6 (MSH6). Reaction products were resolved by SDS-PAGE and analyzed by Western Blot using an antibody against the S6K1 phosphorylation motif (p-Akt substrate, RXXS), or antibodies to detect the recombinant proteins. (c,d) Western blot analysis of HEK293 cells co-transfected with FLAG-MSH6 and S6K1 isoforms immunoprecipitated with anti-Flag beads. Phosphorylation of MSH6 was detected with p-Akt substrate antibody. Quantification of MSH6 phosphorylation is shown in the graph (d). (e,f) Western blot analysis of HEK293 cells co-transfected with HA-Cdk1 and S6K1 with and without UVC 30 J/m² /s (UV) damage isoforms immunoprecipitated with anti-HA beads. Phosphorylation of Cdk1 was detected with p-Akt substrate antibody. Quantification of Cdk1 phosphorylation is shown in the graph (f). The experiment was repeated three times (biological replicate). ** p<0.01 p values were calculated using Student’s t-test (two-tailed).