Fig. 5

TRF1 is phosphorylated in vitro by AKT1. a One μg of purified GST-TRF1 was incubated with 0.2 μg of human GST-AKT1 with or without AKT inhibitor (MK-2206) in kinase buffer containing 5 μCi [γ-³²-P]ATP (left). The peptide RBER-GSK3(14-27) was used as positive controls (right). The reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. b MS/MS spectrum of unmodified TRF1 328-339 (above panel) and of the TRF1 328-339 peptide in which phosphorylation was assigned to T330 residue (below panel). c MS/MS spectrum of unmodified TRF1 246-255 peptide and of the TRF1 246-255 peptide in which phosphorylation was unambiguously assigned to the T248 residue (below panel). Mass error, identification score, and posterior error probability (PEP) are shown for the different peptides. d, e Quantification of TGTLQCETTMER (T330) (d) and of the AATKVVENEK (T248) (e). Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus AKT1. f Schematic representation of TRF1 protein depicting its N-terminal acidic domain, TRFH domain, nuclear localization signal (NLS), and its Myb domain. The residues found phosphorylated by AKT (T248, T330, and S344) were mutated to alanine. g Representative image of in vitro phosphorylation assay of purified GST-TRF1, GST-TRF1(T330A), GST-TRF1 (S344A), GST-TRF1 (T248A), or GST-TRF1 (T330A/S344A/T248A) by GST-AKT1. The reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. As loading control, purified GST-TRF1 protein samples were run in SDS-PAGE and coomassie-stained (below panel). The quantification of mutant P-TRF1 levels normalized to wild-type TRF1 is shown to the right. Student’s t test was used for statistical analysis, P values are shown. Error bars represent standard deviation. n number of independent experiments