Dissociation of PKA-mediated phosphorylation and inactivation of
GSK-3 from a functional PI3K–PKB signaling pathway. (A)
PKA-mediated phosphorylation of GSK-3 is independent of PI3K activity.
After 12–24 h of incubation in serum-free medium, Rat1 and NIH 3T3
cells were stimulated with 8-Br-cAMP (2 mM, 30 min), IGF-1 (75 ng/ml,
10 min, Rat1), or insulin (0.1 μM, 10 min, NIH 3T3) in the absence or
presence of the PI3K inhibitors, wortmannin (125 nM) or LY294002 (12.5
μM). The cells were preincubated with wortmannin or LY294002 for at
least 1 h before addition of 8-Br-cAMP, IGF-1, or insulin.
Phosphorylation of GSK-3α at serine 21 and GSK-3β at serine 9 was
analyzed by immunoblotting with GSK-3 phospho-specific antibodies as in
Fig. 1. (B) Elevation of cAMP levels does not increase
phosphorylation of PKB at serine 473. Rat1 and NIH 3T3 cells were
starved and stimulated with 8-Br-cGMP, 8-Br-cAMP, forskolin,
isoproterenol, IGF, or insulin, as detailed in Fig. 1. PKB
phosphorylation levels were analyzed by immunoblotting with a PKB
phospho-specific antibody that recognizes PKB phosphorylated at serine
473 (New England Biolabs). Reprobing with a PKB antibody reactive with
total PKB (New England Biolabs) shows similar loading among samples.
(C) 8-Br-cAMP or elevation of endogenous
cAMP levels induces no or limited increases in PKB activity. Rat1 and
NIH 3T3 cells were stimulated as in B. PKB activity
toward the crosstide–paramyosin substrate was determined as detailed
in Materials and Methods. The crosstide–paramyosin
substrate phosphorylated at a serine site corresponding to serine
21/9 of GSK-3 is indicated with crosstide-p. The intensities of the
crosstide-p bands were quantified by densitometry. The values beneath
each lane represent relative intensities (%) with bands induced by
IGF-1 in Rat1 cells and by insulin in NIH 3T3 cells defined as 100%.
For comparison with PKB activity, a fraction of each lysate was
analyzed for levels of GSK-3 phosphorylation at serine 21 and 9 by
immunoblotting with GSK-3α and -β phospho-specific antibodies
(Bottom). Data shown are representative of three
independent experiments.