Figure 2.

Serine 224 is required for SRF function. A, SRF^S224A mutant cannot rescue axonal growth deficits of SRF-deficient neurons. P0.5 hippocampal neurons from Srf-NestinCKO mice were transfected with IRES-EGFP empty vector, HA-SRF^WT, or HA-SRF^S224A. Cells were fixed and immunostained for GFP or HA (green) to visualize transfected cells and anti-Tuj1 (red) at 4 DIV. Axonal growth deficits exhibited by SRF-deficient neurons were rescued by SRF^WT alone and not by empty vector or SRF^S224A (arrows). Arrowhead indicates an untransfected cell. Scale bar, 50 μm. B, Quantitation of axon length from A. n = 4 mice; “n” in the bars indicates the number of cells measured for statistics. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). n.s., Not significant. C, SRF^S224A functions as a dominant mutant when expressed in wild-type hippocampal neurons. Cells were transfected with HA-SRF^WT or HA-SRF^S224A and grown for 4 DIV. Cells were fixed and immunostained with anti-HA (green) and anti-Tau (red) antibodies. HA-SRF^S224A expression attenuated axonal growth (arrows). Scale bar, 50 μm. D, Data quantified from C. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). E, Inhibiting GSK-3 activity or mutating serine 224 to alanine abolishes SRF transcriptional activity. HEK293T cells were transfected with 5×-SRE luciferase and Renilla luciferase (for transfection efficiency) and treated with either 6-BIO or cotransfected with control vector, SRF^WT, SRF-S224A, or SRF-VP16. 6-BIO treatment blocked serum-induced SRF-transcriptional activity. Whereas vector and SRF^WT transfected cells showed normal activation of an SRE reporter, the SRF^S224A mutant was severely compromised in activating transcription. Expression of constitutively active SRF-VP16 served as the control. Error bars indicate mean ± SEM.