Neuromodulator-mediated phosphorylation of specific proteins in a neurotumor hybrid cell line (NCB-20).
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.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015762:2-Chloroadenosine; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine; D006822:Hybrid Cells / Q000378:metabolism*; D007118:Immunoassay; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010750:Phosphoproteins; D015762:2-Chloroadenosine; D005576:Colforsin; D012701:Serotonin; D000001:Calcimycin; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D000527:Alprostadil; D000241:Adenosine; D015056:1-Methyl-3-isobutylxanthine