FIG. 3.
Phosphorylation of GSK-3 by cAMP-PKA does not require activation of Akt. (A) Cerebellar granule neurons were cultured for 7 days, washed twice, and placed in serum-free medium containing 5 mM KCl. Two hours later, the cells were either left untreated or treated with 10 μM forskolin for the indicated times. Some neurons were incubated with IGF-I (50 ng/ml) in the absence or presence of wortmannin (wort) (100 nM) for 30 min as positive controls. Cell lysates were immunoblotted with antibodies to phospho-S473 Akt and phospho-T308 Akt. The membrane was then stripped and reprobed with antibodies to phosphorylation-independent Akt. (B) Neurons were treated as described for panel A. Akt was immunoprecipitated from cell lysates with an immobilized Akt antibody, and kinase activity was determined by an in vitro kinase assay using 1 μg of recombinant GSK-3α fusion protein as the substrate. Phosphorylation of GSK-3α was detected by immunoblotting with phosphospecific GSK-3α/β (Ser21 and Ser9) antibody (bottom). The membranes were stripped and reprobed with the antibody to phosphorylation-independent Akt (top). (C) Neurons were transfected with the indicated expression vectors (along with pCMV-β-Gal), and 24 h later they were placed in medium containing 25 mM KCl (K+) plus serum or in deprivation medium (5 mM KCl, no serum) in the presence or absence of 10 μM forskolin (FK), 500 μM CPT-cAMP, or 50 ng/ml of IGF-I per ml. After 20 h, the neurons were fixed and costained with an antibody to β-Gal and Hoechst 33258. Apoptosis was quantified by scoring the percentage of transfected neurons in the adherent cell population with condensed or fragmented nuclei. Data are from three experiments and are the means ± SEMs.