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. 2004 Dec;24(23):10366–10380. doi: 10.1128/MCB.24.23.10366-10380.2004

FIG. 5.

FIG. 5.

(A) Phosphorylation of p53 in Thr18 in vitro by using GST-VRK1 fusion. As substrates in the kinase reaction, GST-p53wt (FP221 fusion protein), GST-p53T18A (amino acids 1 to 85), GST-p53 (amino acids 90 to 290), GST-p53 (amino acids 290 to 390), GST-Mdm2 (amino acids 1 to 188), and GST proteins were used together with 2 μg of GST-VRK1 in a radioactive kinase assay. At the bottom is shown staining of the gel with Coomassie blue. ns, nonspecific band. (B) Effect of p53 phosphorylation in Thr18 interferes with its binding to Mdm2 analyzed in a pull-down assay. The wild-type GST-p53 and its phosphorylated form with VRK1 were mixed with Mdm2 protein that was synthesized and labeled in an in vitro transcription-translation system with [35S]methionine. The bound Mdm2 was detected by its radioactive signal, which was used for quantitation as represented by the bars. The GST-p53 protein loaded in the gel was detected by Coomassie blue staining. Thr18 phosphorylation was detected with the FTP18 monoclonal antibody. (C) H1299 human tumor cells (p53−/−) cotransfected with VRK1 show stabilization of p53, which is partially reverted but not abolished by overexpression of cotransfected Mdm2. H1299 cells were transfected with 1 μg of pCB6+p53 expression plasmid alone or together with plasmids pCOC-Mdm2 (2 μg) and pCDNA-VRK1 (4 μg) where indicated. One microgram of plasmid encoding ubiquitin was added in all cases. Whole-cell extracts were prepared 36 h after transfection and analyzed by Western blotting with the corresponding antibody. (D) The stabilization of p53 by VRK1 is to some extent independent of the presence of Mdm2. Mouse embryo fibroblasts derived from double-knockout mice (p53−/− Mdm2−/−) were cotransfected with the indicated proteins. The cells were processed as for panel C. MG132 proteasome inhibitor was added for 6 h prior to lysis where indicated on the right. (E) Effect of VRK1 on the ubiquitination of p53 by Mdm2. H1299 cells were transfected with the indicated proteins as for panel C, and 25 μM proteasome inhibitor MG132 was added 30 h after transfection for 6 h. Total p53 was immunoprecipitated with the polyclonal antibody CM1 and detected with the monoclonal antibodies DO-1 and Pab1801. Mdm2 protein coimmunoprecipitated and was detected using the 4B2 antibody. The ubiquitinated form of p53 is indicated. IP, immunoprecipitation; IB, immunoblotting.