Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

To define the proteins whose expression is regulated
by cAMP and
protein kinase A (PKA), we used a quantitative proteomics approach
in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma
cells. We also compared the impact of endogenous increases in the
level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor
isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP).
We identified 1056 proteins in WT and kin- S49 cells and found that
8-CPT-cAMP and Fsk with IBMX produced differences in protein expression.
WT S49 cells had a correlation coefficient of 0.41 between DNA microarray
data and the proteomics analysis in cells incubated with 8-CPT-cAMP
for 24 h and a correlation coefficient of 0.42 between the DNA microarray
data obtained at 6 h and the changes in protein expression after incubation
with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher
level of basal expression in kin- S49 cells than in WT cells. Consistent
with this finding, kin- cells are less sensitive to cell killing and
generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad
impact on protein expression in mammalian cells, including in the
regulation of Gsr and oxidative stress.