Figure 7.
mDia1 interfering derivatives block SRF activation and cytoskeletal rearrangements. (A) Interfering derivatives block activation of SRF by serum stimulation. NIH3T3 cells were transfected with SRF reporter and plasmids expressing mDia1 derivatives F1F2Δ1, F1F2Δ2, F1F2ΔC2, or FH3/M (2.0 μg), or wild-type β-actin (1.0 μg), maintained in 0.5% FCS for 36 h, and then stimulated with 15% serum as indicated. Reporter activation is presented as mean ± SEM of three independent experiments. (B) F1F2Δ1 does not block activation of TCF Elk-1 by serum stimulation. NIH3T3 cells were transfected with the LexA operator controlled reporter LexOP-Luc (0.1 μg), an expression plasmids encoding NLex. ElkC (0.1 μg), a chimeric transcription factor comprising the C-terminal activation domain of Elk-1 fused to the bacterial LexA repressor (Marais et al., 1993), and mDia1 derivative F1F2Δ1 (2.0 μg). Cells were serum-stimulated as in A. Reporter activation is presented as mean ± halfrange of two independent experiments. (C) F1F2Δ1 blocks activation of SRF by LPA stimulation. NIH3T3 cells were transfected with SRF reporter and plasmids expressing mDia1 derivative F1F2Δ1 (2.0 μg), or wild-type β-actin (1.0 μg), maintained in 0.5% FCS for 36 h, and then stimulated with 10 μM LPA as indicated. Reporter activation is presented as mean ± SEM of three independent experiments. (D) Src activity is not required for serum-stimulated SRF activation. NIH3T3 cells were transfected with SRF reporter and plasmids expressing C-terminal Src Kinase (CSK; 2.0 μg) or kinase inactive SrcK298 M (SrcKD; 2.0 μg) and processed as in A. Where indicated, cells were pretreated for 1 h before stimulation with the Src inhibitor PP2 (0.25 μM, 1.0 μM). Reporter activation is presented as mean ± SEM of three independent experiments. (E) F1F2Δ1 blocks stress fiber induction by LPA. NIH3T3 cells were transfected with a plasmid expressing mDia1 derivative F1F2Δ1 (2.0 μg), maintained in 0.5% FCS for 36 h, and then stimulated with 10 μM LPA for 2 min before fixation and staining for F-actin and the transfected protein epitope tag. Arrows indicate the transfected cells.