Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells: protein modifications, inductions and repressions.
In this study, we used two-dimensional gel electrophoresis to analyze the responses of cultured S49 mouse lymphoma cells to incubation with analogs or inducers of cyclic AMP (cAMP). Putative phosphorylations were detected by charge alterations in proteins labeled with 35S--methionine and, in some cases, confirmed by labeling with 32P--phosphate. We assessed the relative stabilities of proteins affected by cAMP, the periods of susceptibility of proteins to cAMP-dependent modification and any cAMP-mediated changes in protein synthesis or stability. Five proteins (of about 650 resolved) behave as expected for "orthodox" substrates of a cAMP-activated protein kinase: both newly synthesized and prelabeled forms of these proteins are subject to modification; this modification involves an acidic charge shift of about one unit; and cAMP-mediated conversion of these proteins to their modified forms is virtually complete. The acidic forms of at least three of these proteins also exhibit cAMP-mediated increases in 32P--phosphate incorporation. Each protein comprised less than approximately 0.005% of cellular protein. Under basal conditions they appear to be phosphorylated to an extent about 20--30% of that found in fully stimulated cells. Nine proteins show cAMP-dependent changes in rates of synthesis with six inductions and three repressions. Most of these changes are of a magnitude of about 3 to 5 fold, and reach their maximal extents after about 4--5 hr of exposure to dibutyryl cAMP. In addition to the phosphorylations, inductions and repressions mentioned above, approximately 12 other reproducible cAMP-dependent changes in protein patterns are observed. Mutant cell lines deficient in catalytic activity of cAMP-dependent protein kinase show none of the changes in protein pattern attributable to cAMP.
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D008223:Lymphoma / Q000188:drug therapy / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D009374:Neoplasms, Experimental / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D009363:Neoplasm Proteins; D003994:Bucladesine; D000242:Cyclic AMP; D011494:Protein Kinases