Figure 9. Role of PDK1 in anti-apoptotic activity of the ERK1/2 pathway.

A, Neurons were transfected with equimolar mixes of shERK1-1+shERK1-2 (shERK1) or shERK2-1+shERK2-2 (shERK2). Plasmid dosing and treatments were as described for Fig. 5. BDNF-mediated suppression of TD-induced apoptosis was abolished by knockdown of either ERK. B, Neurons were co-transfected with expression plasmids for β-gal (pON260) and either wild type (wt), dominant negative (dn) or constitutively active (ca) mutant forms of MKK1 (0.2 + 1 µg of plasmid DNA/5×10⁵ neurons, respectively). Empty expression vector (pcDNA3, Vector) was used as a control. After 24 hours, neurons were sham treated (control) or trophic-deprived (TD) for next the 24 hours. The caMKK1 reduced the apoptotic response to TD. C, Neurons were co-transfected with pON260, MKK1ca and the indicated shRNAs (0.2 + 0.8 + 0.2 µg of plasmid DNA/5×10⁵ neurons, respectively). The shERK1/2 or shRSK1/2 consisted of equimolar mixes of shERK1-1 + shERK1-2 + shERK2-1 + shERK2-2 or shRSK1-1 + shRSK1-2 + shRSK2-1 + shRSK2-2, respectively. The empty expression vector (pcDNA3, vector) or shGFP were used as controls for MKK1ca or shRNAs, respectively. Cells were treated as in B. Either shERK1/2 or shRSK1/2 or shPDK1 reduced the caMKK1-mediated neuroprotection against TD indicating critical role of PDK1-RSK1/2 interactions for the survival signaling by the MKK1-ERK1/2 pathway. D, Neurons were co-transfected with pON260, caMKK1 and the activated PDK1A280V mutant (0.2 + 0.2 + 0.4 µg of plasmid DNA/0.5×10⁶ neurons, respectively). The pcDNA3.1 was used as a vector control (Vector). Cells were treated as in B. At the plasmid dosage applied in these experiments, neither caMKK1 nor PDK1A280V alone were sufficient to protect against TD. However, their co-expression suppressed the TD-induced apoptosis indicating the survival synergy of the PDK1 and MKK1-ERK1/2 pathways. In A–D, averages of triplicate determinations from three independent experiments±SEM are shown; **, p<0.01; ***, p<0.001, NS, non-significant. E, Our data supports a model that BDNF- or MKK1ca-mediated neuroprotection against the TD-induced apoptosis employs PDK1-dependent activation of RSK. Also, the survival requirement for the PDK1-RSK signaling suggests that RSK-NTK substrates are critical for the anti-apoptotic neuroprotection.