Proposed role of ATM-mediated I-2 phosphorylation in the activation of PP1 and the signaling cascade in G₂/M checkpoint regulation in response to IR-induced DNA damage. I-2^S43p, I-2 phosphorylated at serine 43; H3 Ser^10p, H3 serine 10 phosphorylation.

The IR-induced dissociation of PP1 and I-2 requires functional ATM. Two hours after IR, immunoprecipitation from nuclear extracts was performed using an anti-PP1 antibody and immunoblot analyses were conducted using antibodies against I-2 or PP1 from HeLa and 293T cells (A), fibroblasts proficient (GM0637) or deficient (GM9607) in ATM (B), and isogenic fibroblasts deficient (PEB-vector) or proficient (PEB-YZ5) in ATM (C).

ATM phosphorylation of I-2 at serine 43 is required for the dephosphorylation of PP1 threonine 320 in response to DNA damage. 293T cells were transiently transfected with an empty vector, Xpress-tagged wild-type I-2 (wt-I-2), or S43A mutant I-2 and treated without or with IR (6 Gy). Nuclear extracts were subjected to immunoblotting using anti-phospho-threonine 320 (PP1-Thr^320p) or anti-PP1 antibody.

ATM is required for IR-induced I-2 serine 43 phosphorylation. Shown are Western blot analyses of nuclear extracts from HeLa cells, 293T cells, EBV-transformed lymphoblast cell lines proficient (GM0536) and deficient (GM1526) in ATM, and SV40-transformed human fibroblast isogenic cell lines PEB-vector (ATM deficient) and PEB-YZ5 (expressing reconstituted ATM) with the phospho-serine 43 antibody (Ser43p).

ATM phosphorylates I-2 at serine 43 in vitro. (A) Immunoprecipitated Flag-tagged wild-type (wt) or kinase-dead (kd) ATM was incubated with recombinant proteins consisting of fusions between GST and peptides derived from various regions of human PP1 or I-2. The positions of the amino acids corresponding to each peptide are indicated at the top. p53 peptides (amino acids 1 to 101 for either the wild-type or the serine 15-to-alanine mutant form) were used as controls. (B) The full-length wild-type or serine 43-to-alanine mutant form of I-2 was used as the substrate for wild-type or kinase-dead ATM for the in vitro kinase assay. (C) Sequence homology of I-2 around serine 43 in different species. Underlining in the sequence from human I-2 indicates a sequence highly conserved among mammalian species.

ATM phosphorylation of I-2 at serine 43 is required for the dissociation of the PP1-I-2 complex and the activation of PP1. 293T cells were transfected with an empty vector or the Xpress-tagged wild-type or S43A mutant form of I-2 and mock treated (0 Gy) or treated with IR (6 Gy). (A) The exogenous I-2 was immunoprecipitated with an anti-Xpress antibody, and the immunoprecipitates (IP) were probed with an anti-PP1 or anti-Xpress antibody. wt-I-2, wild-type I-2; Ser43p, phospho-serine 43 antibody. (B) Endogenous PP1 was immunoprecipitated with anti-PP1, and the immunoprecipitates were probed with anti-PP1 or anti-Xpress antibodies. (C) Endogenous PP1 was immunoprecipitated and subjected to in vitro phosphatase assays. Error bars represent ±1 standard deviation, and the means of results from three independent experiments are graphed.

ATM phosphorylation of I-2 is required for the activation of the G₂/M checkpoint and the inhibition of Aurora-B in response to IR-induced DNA damage. (A) HeLa cells were transfected with an empty vector, Xpress-tagged wild-type I-2 (wt-I2), the S43A mutant form of I-2 (S43A), wild-type ATM (wt-ATM), or kinase-dead ATM (kd-ATM) and treated without IR or with IR (6 Gy). Ninety minutes after IR, cells were harvested and subjected to the flow cytometry-based phospho-histone H3 staining assay. Error bars represent ±1 standard deviation, and the means of results from three independent experiments are graphed. Shown under the bar graph are the Western blot results demonstrating the expression patterns of the exogenous proteins. (B) Cells were treated without or with IR (6 Gy), and Aurora-B was immunoprecipitated and subjected to in vitro kinase assays using histone H3 as the substrate. Phosphorylation signals were quantified by a phosphorimager. (C) HeLa cells were transfected with an empty vector, the Xpress-tagged wild-type or S43A mutant form of I-2, or wild-type or kinase-dead ATM and treated without IR or with IR (6 Gy). The Aurora-B kinase assays were performed 90 min after IR. In panels B and C, values for activity levels are shown relative to the activity level of the control, which was set at 1.