SMC3 is phosphorylated at Ser-1067 and Ser-1083. MS/MS spectra that identify Ser-1067 (A) and Ser-1083 (B) as phosphorylated. SMC3 was immunoprecipitated from nuclear extracts made from HeLa cells and further isolated on SDS-PAGE. The SMC3 band was in-gel digested with trypsin and analyzed with capillary liquid chromatography-MS/MS.

The ATM-NBS1 pathway regulates Ser-1083 phosphorylation of SMC3 in response to IR. A, Western blotting of SMC3 phosphorylation of Ser-1083 in siVimentin- and siATM-transfected in HeLa cells that were irradiated with 10 Gy IR and recovered for 1 h. B, requirement of NBS1 and its phosphorylation for IR-induced phosphorylation of SMC3 at Ser-1083. NBS cells complemented with NBC1 (NBS1-WT) or an empty vector (NBS1-Lbi) were treated with 10 Gy of IR and harvested at the indicated times and Western blotted for SMC3 Ser-1083 phosphorylation. C, in vitro phosphorylation of SMC3 by ATM. FLAG-ATM overexpressed in 293T cells was immunoprecipitated by anti-FLAG antibody and incubated with a GST-SMC3 fragment (amino acids 881–1218) and γ-³²P-labeled ATP. In vitro phosphorylation was detected by autoradiography. The amount of SMC3 fragment on SDS-PAGE gel was shown by Coomassie Brilliant Blue (CBB) staining. KD, kinase-dead.

CK2 is required for phosphorylation of Ser-1067 and regulates Ser-1083 phosphorylation in response to IR and intra-S phase checkpoint. A, CK2 is knocked down by co-transfection of siRNA against both α and α′ subunits. B, quantitative mass spectrometric measurements of Ser-1067 phosphorylation. SMC3 was immunoprecipitated from siCK2 (both CK2α and CK2α′ was knocked down) and siVim (as a control)-transfected cells. The percentage of Ser-1067 phosphorylation was normalized to that in the siVim cell. C, quantitative mass spectrometric measurements of Ser-1083 phosphorylation. SMC3 was purified from siCK2 and siVim cells with or without IR treatment. Phosphorylation of Ser-1083 in indicated conditions was measured and normalized to the level in siVim-transfected cells without IR. D, RDS measurements in siCK2 cells and siVim cells. Sixty hours after siRNA transfection, HeLa cells were labeled with ¹⁴C for 36 h and then treated with 10 Gy IR. RDS was measured at the indicated times.

SMC3 is phosphorylated at Ser-1067 and Ser-1083 in cycling and IR-treated cells. A, characterization of pS1083-SMC3 phospho-specific antibody. V5-SMC3-WT and V5-SMC3-S1083A were transfected in 293T cells, and cells were irradiated with 10 Gy IR. SMC3 was immunoprecipitated (IP) with a V5 antibody and Western blotted with pS1083-SMC3 antibody. B, phosphorylation of the endogenous SMC3 at Ser-1083. HeLa cells were irradiated with 10 Gy of IR and allowed to recover for 1 h. Whole cell lysates were Western blotted with antibodies against pS1083-SMC3 and SMC3. C, quantitative mass spectrometric measurement of Ser-1067 and Ser-1083 phosphorylation induction in response to IR. HeLa cells were labeled with light or heavy isotope and treated with or without IR, respectively. Equal amounts of two isotope-labeled cells were mixed for the subsequent processing. Phosphorylation of Ser-1067 and Ser-1083 in light labeled SMC3 (IR-treated) are normalized to those in heavy labeled SMC3 (untreated) to calculate phosphorylation induced by IR.

Phosphorylation of both Ser-1067 and Ser-1083 is required for intra-S phase checkpoint activation. A, RDS measurements of cells transiently transfected with WT, mutant SMC3, or kinase-dead ATM (ATMKD) genes. Plasmids encoding genes of interest were transfected into 293T cells with Lipofectamine™, and cells were labeled with ¹⁴C 48 h after transfection. Cells were treated with 10 Gy IR and allowed to recover for 1 h. RDS was measured as described under “Materials and Methods.” Western blotting by the V5 antibody indicates the expression levels of exogenous WT and mutant V5-SMC3. B, RDS measurements of FLAG-SMC3 stable cell lines. WT or mutant FLAG-SMC3 cell lines were cultured in Dulbecco's modified Eagle's medium with doxycycline (DOX) for 24 h before ¹⁴C labeling. Cells were treated with 10 Gy IR and allowed to recover for 1 h. RDS was measured as described under “Materials and Methods.” Western blotting by the FLAG antibody was used to determine the expression levels of exogenous WT and mutant FLAG-SMC3 after doxycycline addition for 24 h. AA, S1083A/S1067A. C, SMC3 Ser-1083 phosphorylation in a S1067A stable cell line. FLAG-SMC3-WT and -S1067A-expressing 293T cell lines were treated with 5 Gy of IR and recovered for the indicated times. WT and mutant FLAG-SMC3 were immunoprecipitated (IP) from cells lysates with anti-FLAG M2 antibody and Western blotted with phospho-Ser-1083 or FLAG antibody.