Figure 3. PDK1-mediated activation of RSK1/2 in BDNF-stimulated neurons.

Neurons were stimulated with 10 ng/ml BDNF as indicated. In A–B, western blot analysis of RSK1/2 phsphorylation at Ser221/227 is presented. Blots were re-probed to detect total RSK1/2 expression (RSK1/2). For quantifications, the ratios of pSer221/227 to total RSK1/2 were compared to unstimulated controls; averages of two independent experiments ±SD are presented. A, BDNF increased PDK1-mediated phosphorylation of RSK1/2. B, The BDNF-mediated increase of pSer221/227 required activities of the ERK1/2 pathway and PDK1. While in neurons treated with either the vehicle (0.2% DMSO, Veh) or the 30 µM PI3K inhibitor LY294002 (LY), a 1 hour BDNF treatment elevated pSer221/227, the 50 µM MKK1/2 inhibitor U0126 or the 50 µM PDK1 pathway inhibitor TCPK blocked that effect. Experiments providing additional validation of the inhibitors are presented in Fig. S1B (Supplementary Data). C, Without drug inhibitors (Veh), cortical neurons that were sham-treated (control) or trophic deprived (TD) for 3 hours and then stimulated with BDNF for 1 hour, showed increased RSK1/2 NTK activity as determined by immunocomplex kinase assay. The ERK1/2- or PDK1 pathway inhibition abolished RSK1/2 NTK activation by BDNF. Data represent 2 independent experiments ±SD. D, The co-incidence of the MKK1/2-ERK1/2 and PDK1-RSK1/2 signaling in BDNF stimulated neurons. Representative confocal images of neurons that were co-immunostained for pERK1/2 (pThr183-pTyr185, green) and pSer221/227 (red). BDNF stimulations were performed 1 hour after placing neurons in serum free media supplemented with MK801 to reduce the basal ERK1/2 signaling. Note nuclear localization of PDK1-phosphorylated RSK1/2 in BDNF-treated neurons. Most pERK appeared cytosolic. Similar patterns were observed in 2 independent experiments. Additional data illustrating the co-incidence of the BDNF-mediated RSK1/2 activations at the NTK and CTK are presented in Fig. S2 (Supplementary Data).