FIGURE 3.

TOPK phosphorylates Prx1 (Ser-32). A, comparison of amino acid sequences of Prx1 and Prx3. Amino acids Ser-32 and Ser-126 in Prx1 correspond with Asp-32 and Ala-126 in Prx3, respectively. B, in vitro kinase assay using [γ-³²P]ATP to determine whether TOPK phosphorylates Wt or mutant (S126A or S32A) Prx1. Proteins were visualized by autoradiography. C, TOPK binds with phosphorylated Prx1. TOPK-phosphorylated or nonphosphorylated Prx1 (Wt) or Prx1 mutant (S32A) protein (in vitro kinase assay) were used for binding with GST-TOPK beads. TOPK and Prx1 binding was confirmed by immunoblotting with anti-Prx1. D and E, CD spectra in the far UV range were prepared to determine changes in the secondary structure of Prx1 induced by phosphorylation. D, Prx1-Wt (solid line, 1) or phosphorylated Prx1-Wt (dashed line, 2). E, Prx1-S32A (solid line) or phosphorylated Prx1-S32A (dashed line).