Figure 7

MAG-induced tyrosine phosphorylation of FAK is required for growth cone repulsion to MAG. A-C. MAG induces phosphorylation of FAK. Shown in (A) is the time course of FAK phosphorylation after MAG stimulation (2 μg/ml) of rat hippocampal neurons. Cell lysates were immunoprecipitated with anti-FAK antibodies and immunoblotted with the pY-20 antibody for phosphorylated tyrosine residues. Shown in (B) are experiments in the presence or absence of Ha2/5 (1.0 μg/ml) or echistatin (100 nM). Shown in (C) are experiments with the treatment of WT-MAG (RGD) or mutant MAG (KGE). D. MAG induces phosphorylation of FAK on residues Y397 and Y861. Cell lysates of hippocampal neurons after MAG stimulation were immunoprecipitated with anti-FAK antibodies and immunoblotted with tyrosine phosphorylation site-specific antibodies to FAK. E-G, Phosphorylation of FAK on residues Y397 and Y861 is required for MAG-induced growth cone repulsion. Hippocampal neurons were transfected with expression constructs for GFP, WT-FAK-GFP (E), FAK-Y397/861F-GFP (F), GFP and control shRNA, GFP and shRNAs against FAK. Growth cones of GFP⁺ neurons were examined in a gradient of MAG (150 μg/ml in the pipette). Sample images and traces were shown similarly as in Fig. 2 (A-C). Scale bar: 20 μm for microscopic images and 5 μm for traces. Shown in (G) is the summary of growth cone turning angles. Values represent mean ± s.e.m. Numbers associated with the bar graph indicate the number of growth cones analyzed. "*" indicates significant difference from the control (neurons transfected to express GFP alone; p < 0.01, ANOVA).