Figure 4. Plk4 binds Cdc6 in S phase and phosphorylates Cdc6.

(a) Endogenous Cdc6 interacts with Plk4 in cells. HEK293 total cell extract was immunoprecipitated with a Cdc6 antibody, and probed with Cdc6 and Plk4 antibodies. (b) Plk4 directly binds Cdc6 in vitro. Sepharose beads coupled with purified GST or GST-Plk4 were incubated with purified His-Cdc6 and analysed using His antibody. The loadings of indicated proteins are shown by Coomassie blue staining. (c) The Plk4 kinase-dead mutant K41M does not bind Cdc6 in cells. HEK293 cells were co-transfected with Myc-Cdc6 and GFP-Plk4 WT or K41M mutant. Total cell extracts were immunoprecipitated with GFP antibody, and probed with Myc and GFP antibodies. (d) Cdc6 interacts with Plk4 during S phase. HEK293 cells were co-transfected with GFP-Plk4 and Myc-Cdc6 and synchronized at the G1/S transition. The cells were then released for the indicated time period, collected for immunoprecipitation using GFP antibody, and analysed by western blotting using Myc, GFP, cyclin B and GAPDH antibodies. (e) N-terminus of Cdc6, but not C-terminus of Cdc6, interacts with Plk4. GFP, GFP-tagged Cdc6 WT or Cdc6 truncate was immunoprecipitated with GFP antibody from total cell extract of HEK293 cells co-transfected with Myc-Plk4. Immunoprecipitated proteins were analysed using Myc and GFP antibodies. (f) Cdc6 is phosphorylated on S30 and T527 in cells. HEK293 cells were transfected with GFP-tagged Cdc6 or Cdc6 mutants, and the cell lysates were probed with GFP antibody using Phos-Tag acrylamide assay (upper panel). (g) Plk4 phosphorylates Cdc6 on S30 and T527 in vitro. Purified GST-Plk4 was incubated with purified His-tagged Cdc6 or Cdc6 mutant proteins in the presence of [γ-³²P]-ATP, followed by autoradiography. Coomassie blue staining shows the loading of indicated proteins. Asterisk indicates the phosphorylated Cdc6. (h) Quantitation of (g) for Cdc6 WT, S30A, T527A or 2A phosphorylation from four independent experiments. The relative intensity of the phosphorylation signal of each protein was normalized to its protein level by ImageJ. The phosphorylation signal intensity of Cdc6 WT was arbitrarily set at an intensity of 1.0. The statistical data in h is presented as means±s.d. ***P<0.001 (Student's t-test).