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. 2012 Nov 6;288(5):3359–3369. doi: 10.1074/jbc.M112.384289

FIGURE 5.

FIGURE 5.

PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186. A, PAK6 phosphorylates Mdm2. HEK293 cells were transfected with pcDNA3.1-Myc-His-PAK6 and lysed for immunoprecipitated with anti-Myc antibody; the immunoprecipitated PAK6 kinase was incubated with GST-Mdm2 and analyzed by autoradiography. Histone H3 served as positive control. B, PAK6 phosphorylates Mdm2 between amino acids (aa) 121 and 300. In vitro kinase assay was performed using commercialized PAK6 kinase and GST-Mdm2 full-length and deletions as indicated and analyzed by autoradiography (auto). C, Mdm2 Thr-158 and Ser-186 are the phosphorylation sites by PAK6. In vitro kinase assay was performed using commercialized PAK6 kinase and GST-Mdm2 (121–300 amino acids) or GST-Mdm2 single-site mutants as indicated and analyzed by autoradiography. Histone H3 (H3) served as a positive control.