Sl. Lee et al., METHODS FOR STUDYING SYNTHESIS, TURNOVER, AND PHOSPHORYLATION OF CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE IN MAMMALIAN-CELLS, Molecular and cellular endocrinology, 116(2), 1996, pp. 233-241
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.