Figure 2. Cdk1 specifically phosphorylates unbound TRF1 and this phosphorylation keeps TRF1 free of chromatin.

(a) Two micrograms of recombinant wild type TRF1, mutant TRF1-R425V or H1 was pre-incubated with no DNA or 5 μg of pTH12-NT or pTH12, followed by Cdk1 kinase assays. (b) Differential salt extraction of chromatin on HeLaI.2.11 cells treated with DMSO or nocodazole (Noc). (c) Western analysis. (d) Quantification of total TRF1 and phosphorylated TRF1 from (c). The signals from the western blots were quantified with densitometry. The level of total TRF1 and phosphorylated TRF1 are represented in arbitrary units after their signals were normalized relative to those of γ-tubulin. A Student’s two-tailed t test was used to derive all P values. Standard deviations (SDs) from three independent experiments are indicated. (e) Dot blots of ChIPs with anti-pT371 or anti-TRF1 antibody. HeLaI.2.11 cells were treated with DMSO or nocodazole. Anti-IgG antibody was used as a negative control in this experiment and all the following ChIPs shown in this article. (f) Quantification of ChIPs from (e). ImageQuant analysis was used for quantification in this experiment and all the following ChIPs shown in this article. The P value was calculated using a Student’s two-tailed t test. SDs are derived from at least four independent experiments. (g) Quantification of ChIPs with anti-pT371 or anti-TRF1 antibody from hTERT-RPE cell extracts. The P value was calculated using a Student’s two-tailed t test. SDs from three independent experiments are indicated. (h) DNA binding assays. Prior to gel shift assays, recombinant wild type TRF1 (66 ng) or T371A (85 ng) was incubated with BSA (1 μg) and Cdk1 with or without 5 mM cold ATP. BSA was used as a competitor for the nonspecific activity of Cdk1. (i) Recombinant Plk1 was incubated with recombinant wild type TRF1 or β-casein in the presence of γ-³²P-ATP. Recombinant TRF1 comigrates with Plk1 on an 8% SDS-polyacrylamide gel. (j) Sequential kinase assays. Recombinant wild type TRF1 or TRF1-T371A was incubated with cold ATP with or without Cdk1, followed by addition of γ-³²P-ATP in the presence of Plk1 or no Plk1 as indicated above the lanes.