Fig. 7

Phosphorylation of GEF-H1 by CaMKI increased its GEF activity. a NT-3-induced minor neurite retraction was abolished by knockdown of GEF-H1 (siCont/PBS = 29, siCont/NT-3 = 26, siGEF-H1#2/PBS = 23, siGEF-H1#2/NT-3 = 26 neurites from three independent experiments). b The increase in FRET efficiency in the cell body induced by NT-3 was abolished by knockdown of GEF-H1 (siCont/PBS = 5, siCont/NT-3 = 8, siGEF-H1#2/PBS = 6, siGEF-H1#2/NT-3 = 15 neurons from three independent experiments). c CaMKI increased the GEF activity of GEF-H1. GEF-H1 was co-transfected with myc-CaMKI-WT, -CA or –KD into COS-7 cells. The cell extracts were incubated with GST-RhoA-G17A-bound beads. Active GEF-H1 was pulled down and detected by immunoblotting with anti-myc (top). Total GEF-H1 and CaMKI were detected by immunoblotting with anti-myc (middle and bottom, respectively). d Bar graphs indicate the ratio of GEF-H1 activity. e The phospho-mimic mutant GEF-H1 exhibited an increment in GEF activity. Cell lysates expressing GEF-H1–WT, –T103A or –T103E were incubated with GST-RhoA-G17A-bound beads. Active (top) or total (bottom) GEF-H1 was detected by immunoblotting with an anti-myc antibody. f Bar graphs indicate the ratio of GEF-H1 activity. g myc-GST (Cont), myc-GEF-H1–WT, –T103A or –T103E was transfected into neurons at 3 DIV. Representative images of the neurons at 4 DIV are shown. Scale bars represent 100 μm. h The percentages of neurons with multiple Tau-1-positive axons. i Bar graphs indicate the ratio of multiple axonal lengths. j, k The length of the longest neurite i and the total neurite length j were determined (GST = 86, GEF-H1 WT = 90, GEF-H1 T103E = 88, GEF-H1 T103A = 90 neurons from three independent experiments). Error bars represent SEM. *P < 0.05 and **P < 0.01