The expression of a
adenosine cyclic 3':5'-monophosphate (cAMP)-
binding protein, regulatory subunit of the type I
cAMP-dependent protein kinase (Rl), and its functional significance in the differentiation of N-18 mouse
neuroblastoma cells were examined.
8-Azidoadenosine cyclic 3':5'-[32P]monophosphate, a photoaffinity-labeling analog of cAMP, and high-resolution
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis were used to identify and quantitate cAMP-
binding proteins in
cell extracts. The induction of differentiation of N-18 mouse
neuroblastoma cells, initiated either by adding dibutyryl
adenosine cyclic 3':5'-monophosphate to the growth medium or by culturing cells in medium supplemented with 1%
fetal calf serum, led to a 3-fold increase in the amount of
8-azidoadenosine cyclic 3':5'-[32P]monophosphate incorporated into Rl, when assayed in vitro. This increased incorporation was attributable to an increase in the amount of Rl rather than to an increase in the affinity of Rl for
8-azidoadenosine cyclic 4':5'-[32P]monophosphate. The subunit molecular weight, isoelectric point, and immunoreactivity of Rl were found to be identical to that of the regulatory subunit of the type I
cAMP-dependent protein kinase purified from bovine skeletal muscle. The increase in Rl was not accompanied by an increase in the
cAMP-dependent protein kinase activity.
DEAE-cellulose column chromatography confirmed the induction of Rl as a free cAMP-
binding protein in the differentiated
neuroblastoma cells. The possibility of a growth-dependent regulation of Rl was also examined. Addition of 2%
dimethyl sulfoxide to cultures of N-18 mouse
neuroblastoma cells inhibited cell growth without increasing the specific activity of Rl.
Dimethyl sulfoxide had no effect on neurite outgrowth or
acetylcholinesterase activity, two parameters characteristic of differentiated cells. The fact that the induction of Rl coincided with differentiation of the
neuroblastoma cells suggests that the expression of Rl may be used as a biochemical index of differentiation in these cells. The presence of a free cAMP-
binding protein, not associated with
cAMP-dependent protein kinase in
neuroblastoma cells, raises important considerations concerning the action of cAMP in the regulation of growth and differentiation.