A PDK-1 allosteric agonist neutralizes insulin signaling derangements and beta-amyloid toxicity in neuronal cells and in vitro
Fig 4
PS48 partly restores inhibited Akt phosphorylation (Akt activation) and GSK3β phosphorylation (Akt activity).
In vitro additions of recombinant PDK (5 ng; 4A.) or pre-immunoprecipitated PDK-1 (4B.). 4A. Aβ peptide addition near completely results in Akt dephosphorylation (lane 3). PS48 added after Aβ peptide (post) in the reaction mixture completely reverses the loss of Akt phosphorylation (lane 5 vs. 3). When pre-added (pre), PS48 partially restores pAkt levels (lane 4). Note commercial recombinant Akt lots come variably phosphorylated (lane 1), but lack enzymatic activity in control reactions until PDK is added (see Figs 4B and 6B and text) and also undergo further phosphorylation. 4B. Restoration of Akt-catalyzed phosphorylation of GSK3β peptide (lanes 5 and 6 vs. 4). 4C. Quantified trend to reverse inhibited Akt activation and activity by PS48 in the presence of 10 μM Aβ42 oligomers (ADDLs). PS48 is active in the 10 nM range. Data from pAktT308, pAktS473 and pGSK3α/β S9 experiments were normalized and pooled (n = 3–6 ea.). ’Fraction of control Akt activation’ is the [Aβ (± PS48 presence) /Control] densitometry signals ratio, where 1.0 represents full restoration.* p < .02, t-test, Aβ 10μM/0μM PS48 (n = 6) vs. combined 100 nM and ≥ 1 μM (1, 10 and 100 μM) data (n = 8). The mean difference is 0.23 ± 0.08, 95% CI = [0.41 to 0.05].