Figure 1.

dPER(ΔC4) is defective in transcriptional repression and DBT-dependent progressive phosphorylation in S2 cells. (A) Schematic diagram of functional domains in the C-terminal half of dPER protein (accession no. P07633). Horizontal lines denote regions internally deleted from full length dPER to generate mutants analyzed in this study. Domains are as follows: Thr-Gly repeats (dark grey box with letter “TG”); dCLK:CYC inhibitory domain (CCID; aa 764 to 1034; white box) (Chang and Reppert, 2003); dPER DBT Binding Domain (dPDBD; aa 755 to 809; bracket) (Kim et al., 2007); putative nuclear localization sequence (aa 813 to 840, hatched box) (Chang and Reppert, 2003). (B) S2 cells were transiently transfected in the presence (+) or absence of (-) pMT-dClk-V5. In addition, some cells were co-transfected with different versions of pAct-dper, as indicated. Shown are the average values from three independent experiments for relative luciferase activity. Luc activity in the absence of pMT-dClk-V5 was set to 1, and all other values were normalized. (C -E) S2 cells were transiently transfected with different versions of V5-tagged pAct-dper (C, E) or non-tagged pAct-dper (D), as indicated (top of panels). In addition, some cells were co-transfected with pMT-dbt-V5, as indicated (+). Exogenous DBT was induced 36 hr after transfection by adding 500 μM CuSO₄ to the medium. Cells were harvested at the indicated times (C), 24 hr (D), or 36 hr (E) after induction, and protein extracts were either directly analyzed by immunoblotting (C - E) or first subjected to immunoprecipitation in the presence of anti-V5 antibodies (D, top panel). Arrowheads indicate hypo-phosphorylated isoforms in each variants of dPER. (D) ‘Input’ indicates protein lysates used for immunoprecipitation. dPER was visualized with anti-dPER antibodies, whereas recombinant DBT with anti-V5 antibodies.