Neuromodulator‐Mediated Phosphorylation of Specific Proteins in a Neurotumor Hybrid Cell Line (NCB‐20)

Publisher Website
Google Scholar
Abstract
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 immu‐nological 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.