Figure 3.
S957 and S966 of SMC1 are phosphorylated in vivo in response to IR. SMC1 was immunoprecipitated from HeLa NE prepared from cells that were irradiated with 20 Gy of IR, recovered for 2 h, and resolved on a 6% SDS-PAGE gel. The top and bottom bands of SMC1 were cut out and analyzed separately. Identification of phosphopeptides was carried out as described using mass spectrometry (Zhang et al. 1998). (a) The MS/MS spectrum of the tryptic phosphopeptide amino acids 945–968 of SMC1 from the bottom band. The spectrum identifies the site of phosphorylation as S957. (b) A portion of the MALDI-TOF spectra of the Asp-N digests of the top band of SMC1 that were treated with (bottom panel) and without (top panel) calf intestine phosphatase (CIP). The arrow marks the phosphopeptide, in which S966 is tentatively assigned as the site of phosphorylation. (c) Test of the specificity of phospho-specific antibodies. Flag-SMC1 WT, S957A, S966A, and S957A/S966A were expressed in 293T cells. Cells were treated with 10 Gy of IR and allowed to recover for 1 h. Flag-SMC1 proteins were immunoprecipitated with a Flag antibody, and Western blotted with phospho-specific antibodies against pS957 and pS966. (d) In vivo phosphorylation of S957 and S966 of SMC1 in response to IR. Untreated and IR-treated 293T cells were analyzed by Western blotting using phospho-specific antibodies against pS957 and pS966. (e) In vitro phosphorylation of SMC1 by ATM. A GST–SMC1 fragment (amino acids 890–1233) and Flag-tagged ATM (wild-type or kinase dead) immunoprecipitated by Flag antibody from transiently transfected 293T cells following 20 Gy of IR and 1 h of recovery were used to perform the kinase assay.