Mouse
neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular
cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]
orthophosphate under conditions where
2-chloroadenosine gave maximum increases of 32P incorporation into
tyrosine hydroxylase in
nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (
pheochromocytoma) cells. When NCB-20 cells were exposed to activators [
5-hydroxytryptamine (5-HT),
prostaglandin E1, or
forskolin], resulting in activation of
cyclic AMP-dependent protein kinase, increased 32P incorporation into two major
proteins [130 kilodaltons (kDa) and 90 kDa] occurred.
5-HT (in the presence of
phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and
forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa
protein. [D-Ala2,D-Leu5]-
enkephalin, which decreased
cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of
tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa
protein in NCB-20 cells. Pretreatment of NCB-20 cells with a
calcium ionophore,
A23187, gave increased phosphorylation of the 90- and 130-kDa
proteins, but
phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (
tumor promoting agent), cell depolarization with high K+, or pretreatment with
dibutyryl cyclic GMP had no effect on phosphorylation of these
proteins. In contrast, phosphorylation of an 80-kDa
protein was decreased by
forskolin, but increased following activation of the
calcium/
phospholipid-dependent
kinase with
tumor promoting agent. Neither the 90-kDa nor the 80-kDa
protein showed any immunological cross-reactivity with
synapsin, a major synaptic
protein known to be phosphorylated by
cyclic AMP-dependent protein kinase and
calcium/calmodulin-dependent protein kinase, but not
calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique
proteins can be phosphorylated by
cyclic AMP-dependent protein kinase in response to hormonal elevation of
cyclic AMP levels. In contrast, an 80-kDa
protein is the primary substrate for
calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate
cyclic AMP levels and thereby activate
cyclic AMP-dependent protein kinase.