Phosphorylation of the Actin Binding Protein Drebrin at S647 Is Regulated by Neuronal Activity and PTEN
Figure 4
pS647-Drebrin is de-phosphorylated in dependence of the PTEN phosphatase activity, but independent of PI3K signalling.
(A) Alignment of S647 DBN. DBN comprises of an N-terminal ADF/cofilin homology domain (ADF), a coiled-coil (C–C) domain, a proline-rich stretch (P) and an unstructured C-terminus. (B) Adult rat brain homogenate was analysed by western blot analysis and probed with anti-DBN antibody or with the affinity purified pS647-DBN antibody. (C) Brain homogenate was incubated on ice or at 37°C for 1 hour to activate endogenous phosphatases, before western blotting and analyses. (D) Cortical neurons were nucleofected with control or DBN specific shRNA. Neuronal lysate was analysed at 3 DIV. (E) Bacterially expressed and purified His-DBN was probed for anti-DBN and anti-pS647-DBN antibodies. (F) Cell lysates prepared from YFP-DBN or YFP-S647A-DBN expressing HEK293 cells were analysed by western blotting using indicated antibodies. (G) 19 DIV hippocampal neurons were stained with pan-drebin (mouse) and pS647-DBN antibodies (rabbit) in the presence of control serum or with the pS647-DBN peptide. Scale bar: 10 µm. (H) GFP-PTEN or the GFP-PTEN CS mutant was co-expressed with Flag-DBN in U87MG cells. Bar graph represents the average band density of pS647-DBN/DBN in 3 independent experiments + sem. *p<0.05. (I) Flag-DBN expressing U87MG cells were treated with wortmannin (wm) for 1 hour at 100 nM. (J) As in (H), but cells were co-transfected with Flag-DBN and GFP-PTEN, or with Flag-DBN and GFP-PTENΔD. Bar graph (right) represents the average band density of pS647-DBN/DBN in 3 independent experiments + sem. *p<0.05.