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 ha s been difficult to monitor their expression and virtually impossible to quantify their synthesis, phosphorylation, and turnover in intact m ammalian cells. We now describe sensitive and quantitative immunochemi cal methods using a goat antibody raised against the recombinant C alp ha isoform of murine C subunit that enable studies of the expression a nd 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 subun its from rat, hamster, and human cell lines, so it should have widespr ead utility. Immunoreactivity with bovine heart C subunit was substant ially weaker. For quantitation of C subunit radioactivity in extracts of cells labeled metabolically with [S-35]methionine, we developed a t wo-cycle immunoadsorption protocol that reduces nonspecific adsorption to negligible levels. A tritium-labeled, truncated C subunit marker p rotein is added to extracts as an internal marker to monitor C subunit recoveries in different samples. For analysis of expression of C subu nit isoforms in different cells or tissues, we describe a nonradioacti ve Western immunoblot procedure that can quantitate C subunit in amoun ts as low as 12 pg. Using extraction conditions that either stabilize or destabilize the phosphate on Thr-197, we show how the relative expr ession and phosphorylation of C alpha and C beta isoforms can be estim ated from SDS-gel patterns resulting from either immunoblot or immunoa dsorption procedures.