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

Abstract

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.

Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A

“…6, a and b), having similar volume density, number and size of mitochondria, and cristae abundance (Table 3), consistent with the health and protection from damage by the reactive oxygen species of kin Ϫ cells (15). These data also suggest that the decreased rates of maximal respiration in kin Ϫ cells (Figs.…”

“…The current mitoproteomic analysis identified 2013 proteins, whereas our previous proteomic study of lysates from WT and kin Ϫ S49 cells identified 1056 proteins (15); 469 proteins were in common between these studies. The greater number of proteins identified in this study may result from the use of iTRAQ 4-plex in place of 8-plex (51).…”

“…Proteomic Analysis-Equal (100 g) aliquots of proteins from WT and kin Ϫ S49 cells (0, 6, and 16 h CPT-cAMP treatment) were prepared for isobaric tagging and analyzed by mass spectrometry (MS) as previously described (15) with the following modification; the peptides were labeled with different 4-plex isobaric tagging for relative and absolute quantitation (iTRAQ) reagents (20). Spectrum Mill v3.03 was used to analyze the MS data as described (15) using 3 biological replicates to calculate protein iTRAQ reporter ion intensities.…”

“…We previously found (14,15) that cAMP acts via PKA to alter the expression of many mRNAs in WT, but not kin Ϫ S49 cells, indicating that PKA produces substantial transcriptional regulation (14). Changes in mRNA may not, however, predict effects on protein expression as mRNA and protein expression may not highly correlate (16,17).…”

Mechanisms of cyclic AMP/protein kinase A- and glucocorticoid-mediated apoptosis using S49 lymphoma cells as a model system

“…cAMP/PKA signaling in immature T cells is apoptotic but has other functional roles, including in metabolism and stress responses. Because kin -cells are devoid of cAMP/PKA signaling, they respond differently to oxidative and metabolic stress (50,51).…”