Figure 8. Requirement of PDK1 for the anti-apoptotic activity of the overexpressed RSK1.

A, After 3 hours of TD, cortical neurons were stimulated with 0 or 2 ng/ml BDNF for 1 hr. At that low concentration, BDNF increased pSer-221/227 levels suggesting RSK1/2 activation. Numbers under the blots indicate the relative levels of pSer-221/227 after normalization against total RSK1/2 (fold of control). B, Neurons were co-transfected with expression plasmids for β-gal (pON260) or the wild type RSK1 (wtRSK1) together with shGFP or shPDK1 as indicated (0.2 + 0.4 + 0.4 µg of plasmid DNA/0.5×10⁶ neurons, respectively). An empty expression vector (pcDNA3, Vector) was used as a control for wtRSK1. After 24 hours, neurons were trophic-deprived for the next 24 hours in the presence of 2 ng/ml BDNF. Combining this low concentration of the neurotrophin together with wtRSK1 protected against the TD induced apoptosis. The protection was PDK1-dependent. C, Neurons were co-transfected with expression plasmids for β-gal (pON260), the constitutively active RSK1 (caRSK1) and the activated PDK1A280V mutant as indicated (0.2 + 0.4 + 0.4 µg of plasmid DNA/0.5×10⁶ neurons, respectively). Empty expression vector (pcDNA3, Vector) was used as a control. At 24 hours post-transfection, cells were TD-treated for the next 24 hours. At the low plasmid dosage used for these experiments, the PDK1A280V was unable to suppress the TD-induced apoptosis (p>0.05). However, co-expression of caRSK1, and PDK1A280V resulted in neuroprotection. Results presented in A, were replicated in two independent experiments. In B–C, data represent averages of triplicate determinations from 3 independent experiments ±SEM; * p<0.05; ** p<0.01; ***, p<0.001; NS, p>0.05.