Fig 2: Validation of phospho-specific Mcl-1 antibody, kinetics of Mcl-1 phosphorylation in response to vinblastine, and effect of MG132A. HeLa cells were untreated or treated with vinblastine (VBL) as indicated and whole cell extracts (WCE, lanes 1 and 2) or immunoprecipitated (IP) Mcl-1 (lanes 3 and 4) subjected to immunoblotting for Mcl-1 (upper panel) or with a phospho-specific antibody (p-Ab) originally developed to detect phospho-Bcl-xL (middle panel) or with a phosphorylation-independent Bcl-xL antibody (lower panel). B. HeLa cells were synchronized at the G1/S boundary by double thymidine block and were either untreated or treated with 30 nM vinblastine (VBL) 1 h after release, then harvested at the indicated times. In the untreated group, time points corresponding to late G2-M and G1 phases are indicated. Extracts were subjected to immunoblotting for the proteins indicated, with GAPDH used as a loading control. Phosphorylated Mcl-1 (P-Mcl-1) was detected with an antibody developed to detect phosphorylated Bcl-xL as in Figure 1A. C. HeLa cells were treated with 30 nM vinblastine (VBL) for 24 h, in the presence or absence of 20 μM MG132 added 4 h prior to harvest, and extracts prepared and subjected to immunoblotting for Mcl-1. In the left panel (Mcl-1 Ab-1), Mcl-1 antibody sc-12756 was used, and in the right panel (Mcl-1 Ab-2), Mcl-1 antibody sc-20679 was used.

Fig 3: Phosphorylation of Mcl-1 on T92 by Cdk1 and analysis of Mcl-1 harboring mutations of Cdk consensus sitesA. His-tagged recombinant Mcl-1 was incubated under phosphorylation conditions with [γ-32P]ATP either in the absence or presence of purified active Cdk1/cyclin A2, as indicated, as described in Materials and Methods. Samples were resolved by SDS-PAGE and stained (Coomassie) and subjected to phosphor-image analysis (32P), as indicated. B. Identification of major phosphorylation site as T92. MS/MS spectrum of phosphorylated Mcl-1 peptide VARPPPIGAEVPDVTApTPAR (2093.07 Da monoisotopic molecular weight) with prominent b and y ions and neutral loss product indicated. The peak at 998.88 m/z is consistent with neutral loss of 79.97 Da characteristic of phosphorylation. The b16, b17-98, and b17 ions shown in the detail from 1550-1800 m/z indicate phosphorylation of Thr92. C, D. HeLa cells stably overexpressing wild-type (WT) or T92A (C) or 5A (D) Mcl-1 were untreated or treated with 30 nM vinblastine (VBL) for 24 h, and extracts immunoblotted for the proteins indicated. Parental HeLa cells are shown in the left lanes of each panel; endogenous Mcl-1 is only weakly detected under these conditions. GAPDH was used as a loading control.

Fig 4: Caspase-3 activation in HeLa cells expressing Mcl-1-9AHeLa cells expressing wild-type (WT) or Mcl-1-9A were treated with 30 nM vinblastine (VBL) for the indicated times, and extracts subjected to caspase-3 assay, as described in Materials and Methods. Results are shown as fold-activation relative to untreated cells, and represent mean ± S.D. (n = 6).

Fig 5: Mcl-1 phosphorylation sites associated with mitotic arrestA. Immuno-purified Mcl-1 was digested with trypsin and peptides were analyzed by MS/MS, as described in Methods and Materials and Supplementary Figure S2, which identified the peptide sequences shown in bold representing 63% coverage. Sites of phosphorylation are underlined. B. Sites of phosphorylation, sequence context, and predicted kinases based on matches to established recognition motifs. Sites marked with an asterisk were manually validated. See text for details.