Methods for studying synthesis, turnover, and phosphorylation of catalytic subunit of cAMP-dependent protein kinase in mammalian cells.
Because of the low abundance of the two major isoforms (C alpha and C beta) of catalytic (C) subunit of cAMP-dependent protein kinase, it has been difficult to monitor their expression and virtually impossible to quantify their synthesis, phosphorylation, and turnover in intact mammalian cells. We now describe sensitive and quantitative immunochemical methods using a goat antibody raised against the recombinant C alpha isoform of murine C subunit that enable studies of the expression and metabolism of C subunit in cultured cells. The antibody reacts well with C alpha and C beta isoforms of murine C subunit and with C subunits from rat, hamster, and human cell lines, so it should have widespread utility. Immunoreactivity with bovine heart C subunit was substantially weaker. For quantitation of C subunit radioactivity in extracts of cells labeled metabolically with [35S]methionine, we developed a two-cycle immunoadsorption protocol that reduces nonspecific adsorption to negligible levels. A tritium-labeled, truncated C subunit marker protein is added to extracts as an internal marker to monitor C subunit recoveries in different samples. For analysis of expression of C subunit isoforms in different cells or tissues, we describe a nonradioactive Western immunoblot procedure that can quantitate C subunit in amounts as low as 12 pg. Using extraction conditions that either stabilize or destabilize the phosphate on Thr-197, we show how the relative expression and phosphorylation of C alpha and C beta isoforms can be estimated from SDS-gel patterns resulting from either immunoblot or immunoadsorption procedures.
Lee
S L
SL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Gorman
K B
KB
Steinberg
R A
RA
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements; D051379:Mice; D010766:Phosphorylation; D051381:Rats
D007527:Isoenzymes; D004492:Edetic Acid; D017868:Cyclic AMP-Dependent Protein Kinases