Fig. 2. The DAD of Fhos in the C-terminus appears to regulate the Fhos activity by interacting with the N-terminus. (A) HeLa cells were transfected with vectors encoding the indicated GFP-fused C-terminally truncation mutants of Fhos. Cells were fixed and then detected by GFP fluorescence (upper panels) or phalloidin staining (lower panels). Bar, 20 µm. (B) The C-terminally truncated mutant proteins are illustrated on the left, and the effects of each mutant on the induction of actin stress fibers (shown in Fig. 2A) are summarized on the right (indicated by plus and minus signs). Asterisks in the DAD indicate the polybasic region (amino acid sequences are shown in Fig. 2C). (C) Sequence alignment of the DAD and the polybasic region of Fhos, Diaphanous, p140mDia, Bni1 and SepA. Identical residues are shown on a black background and similar residues are shown on a gray background. The polybasic region is shaded in dark gray and basic residues are indicated by white letters. (D) The direct interaction between the N-terminus and DAD of Fhos. His–Fhos-N (1-569) or His–Fhos-FH1FH2 (533-1053) was incubated with GST–Fhos-DAD (1081-1145). Proteins were pulled down with glutathione-Sepharose-4B, subjected to SDS-PAGE, and stained with CBB. (E) Role for the polybasic region of the DAD in the intramolecular interaction. His–Fhos-N (1-569) binds directly to GST–Fhos-DAD (1081-1145), whereas it failed to bind to GST-Fhos (1081-1120), which lacks the polybasic region, or GST alone. An in vitro pull-down binding assay was performed as in Fig. 2D.

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