Fig. 2.
dbth andDbtg mutations alter conserved amino acids and strongly reduce kinase activity. A, DBT and HRR25 sequences were aligned using CLUSTAL W algorithm of the MacVector Package. dbth mutation alters Thr44 to Ile (*), andDbtg mutation alters Arg127 to His (#). Sequence numbering is based on GenBank accession numbers O76324 for DBT and AAA34687 for HRR25. Only the kinase domains are shown in the alignment. The homology outside of the kinase domain is not significant. The darkly shaded areas indicate identities, and the lightly shaded areas indicate similarities. B, C,35S-Labeled in vitro-translated HRR25 (B) or DBT (C) were incubated with increasing concentrations of GST-PER 1–640 immobilized on glutathione beads for 3 hr. GST-PER concentrations used were 10, 50, and 200 nm. The beads were pelleted, washed three times, loaded on 10% SDS-PAGE, and autoradiographed. GST was used as a negative control. D, Two different concentrations of GST-PER 1–640, 10 and 100 nm, were immobilized on glutathione beads and incubated with HRR25 and γ-32P for 30 min. The beads were pelleted, washed, loaded on 10% SDS-PAGE, and autoradiographed. GST was used as a control.E, Kinase activity of wild-type and mutant HRR25 proteins was determined by measuring the incorporation of32P from [γ-32P]ATP into AHALS(P)VASLPGLKKK. Kinase activity relative to that of wild-type HRR25 (set to 1) is plotted. Error bars indicate SD.