Figure 5.
GSK3β phosphorylates DCX at Ser327. A, Sequential in vitro kinase assay. Bacterially purified GST–DCX bound to glutathione beads was first primed with wild-type (WT) or kinase dead (KD) FLAG–JNK1 purified from HEK293T cells. After high-salt washes to remove JNK, GST–DCX was subjected to a kinase assay with GSK3β and radiolabeled ATP. The top panel shows 32P incorporation, and the bottom panel shows Coommassie blue staining. Asterisk denotes a likely degradation product of GST–DCX. GSK3β robustly phosphorylates DCX in vitro. B, Alignment of human, rat, and mouse DCX protein sequences in the vicinity of a candidate GSK3 phosphorylation site corresponding to human DCX Ser327. C, In vitro GSK3β kinase assay for JNK-primed WT or S327A GST–DCX, performed as in A. The top panel shows 32P incorporation, and the bottom panel shows Coommassie blue staining. Asterisk denotes a likely degradation product of GST–DCX. D, Lysates of HEK293T cells transfected with the WT HA–GSK3β, kinase-dead HA–GSK3β, or control vector and FLAG–DCX expression plasmids together with the GFP expression plasmid were immunoblotted using the FLAG, HA, and GFP antibodies. E, Lysates of HEK293T cells transfected with the WT HA–GSK3β or control vector and the WT FLAG–DCX or S327A FLAG–DCX expression plasmids were immunoblotted using the p-DCX, FLAG, and HA antibodies. GSK3β induces the phosphorylation of DCX at Ser327 in cells. F, Granule neuron lysates treated with or without lambda phosphatase were immunoblotted using the DCX, phospho-AKT, and AKT antibodies, the last two serving as controls for the phosphatase treatment and loading, respectively. G, Lysates of granule neurons treated for 24 h with 2 mm NaCl (Control) or 2 mm LiCl were immunoblotted using the DCX and 14-3-3β antibodies. H, Lysates of granule neurons treated for 56 h with 2 mm NaCl (Control) or 2 mm LiCl were immunoblotted using the p-DCX and 14-3-3β antibodies. Endogenous DCX is phosphorylated at Ser327 in a GSK3β-dependent manner in neurons. I, Lysates of granule neurons treated for 16 h with 200 nm BIO, a selective inhibitor of GSK3, or the vehicle control DMSO were immunoblotted using the p-DCX, DCX, and HSP60 antibodies. Endogenous DCX is phosphorylated at Ser327 in a GSK3β-dependent manner in neurons.