Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)
Figure 5
Eps8 is phosphorylated by MAPK.
(A) 2-D immunoblot analysis of Eps8 from synaptosomes mock-treated (CTR) or treated with BDNF (BDNF) or pretreated with PD98059 before BDNF stimulation (PD+BDNF), or treated with BDNF followed by incubation with alkaline phosphatase (BDNF+AP). Note the appearance, upon BDNF application, of additional and more intense Eps8 immunopositive spots with more acidic isoelectric points (spots: 6–9). These spots disappear by preincubating synaptosomes with alkaline phosphatase. Insets show the analysis of synaptosomes treated as above, resolved by monodimensional SDS-PAGE and immunoblotted with anti–phospho-MAPK (upper band) or anti-ribophorin (lower band), the latter used as loading control. (B) 2-D immunoblot analysis of Eps8 in GCPs in the absence (CTR) or the presence of BDNF. (C) Recombinant purified His-Eps8 (1–821) or recombinant GST-fused Eps8 fragments (5 µg) were incubated with 10 µCi [γ-32P]-ATP in the presence (+) or absence (−) of purified MAPK (20 ng) in kinase buffer (see Materials and Methods), resolved by SDS-PAGE, stained with Coomassie Blue (lower panel), and subjected to autoradiography (upper panel). The red arrow indicates the shift of Eps8 due to phosphorylation. (D) Recombinant purified GST-Eps8 535–821 wild-type (WT) or S624A, T628A (SATA) mutant (5 µg) were incubated with 10 µCi [γ-32P]-ATP in the presence (+) or absence (−) of purified MAPK (20 ng) in kinase buffer (MB), resolved by SDS-PAGE, stained with Coomassie Blue (lower panel) and subjected to autoradiography (upper panel). In (C and D), MBP (myelin basic protein) was used as a control substrate.