Fig. 6
From: Discovery of long-range inhibitory signaling to ensure single axon formation
GEF-H1 is identified as a new substrate of CaMKI in the brain. a The domain structures of GEF-H1 and its various fragments are represented. b Direct phosphorylation of GEF-H1 by CaMKI. Each purified fragment of GEF-H1 was incubated with recombinant CaMKI–cat in the presence of [γ-32P]ATP in vitro. Asterisks indicate intact GST-fusion proteins. c Phosphorylation of GEF-H1 at Thr103 by CaMKI. Each purified Ala mutant of GEF-H1 was incubated with recombinant CaMKI–cat in the presence of [γ-32P]ATP in vitro. Asterisks indicate intact GST-fusion proteins. d, e Phosphorylation of GEF-H1 in COS-7 cells. GEF-H1 was co-transfected with myc-CaMKI-WT, -constitutive active form (CA) or -kinase-dead form (KD) into COS-7 cells. Cell lysates were analyzed by immunoblotting with anti-pT103, anti-myc, and anti-tubulin antibodies. f, g Phosphorylation of endogenous GEF-H1 in hippocampal neurons. Hippocampal neurons were treated with NT-3 with or without KN-93. Cell lysates were analyzed by immunoblotting with anti-pT103 and anti-GEF-H1 antibodies. Error bars represent SEM. *P < 0.05 and **P < 0.01
