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. 2002 Sep;22(17):6209–6221. doi: 10.1128/MCB.22.17.6209-6221.2002

FIG. 4.

FIG. 4.

Plk phosphorylates TCTP on two serine residues. (A) Both Plk and mitotic lysates phosphorylate TCTP on serine residues. Partial acid hydrolysis followed by phosphoamino acid analysis was performed on GST-TCTP phosphorylated by GST-Plk purified from baculovirus-infected Sf9 cells, immunoprecipitated Plk from nocodazole-treated MEL cells, and crude lysate from nocodazole-arrested HeLa cells. (B) Deletion mapping of in vitro Plk phosphorylation sites in TCTP indicates more than one phosphorylation site. (C) Serine-to-alanine point mutant analysis indicates that Plk phosphorylates TCTP on serines 46 and 64 in a hierarchical fashion. GST-TCTP and mutant fusion proteins were phosphorylated by Sf9-produced GST-Plk in separate in vitro kinase reactions. (D) Serine-to-threonine mutations in TCTP support the two-Plk-phosphorylation-site hypothesis. TCTP mutants with combinations of serines and threonines at resides 46 and 64 were phosphorylated by GST-Plk and subjected to phosphoamino acid analysis. The lack of apparent Plk phosphorylation on the Ser64Thr residue indicates a specificity for serine at this site.