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Neuromodulator-mediated phosphorylation of specific proteins in a neurotumor hybrid cell line (NCB-20).

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 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.
AuthorsE Berry-Kravis, B I Kazmierczak, V Derechin, G Dawson
JournalJournal of neurochemistry (J Neurochem) Vol. 50 Issue 4 Pg. 1287-96 (Apr 1988) ISSN: 0022-3042 [Print] UNITED STATES
PMID2450174 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • Nerve Tissue Proteins
  • Neurotransmitter Agents
  • Phosphoproteins
  • 2-Chloroadenosine
  • Colforsin
  • Serotonin
  • Calcimycin
  • Enkephalin, Leucine
  • Enkephalin, Leucine-2-Alanine
  • Cyclic AMP
  • Tyrosine 3-Monooxygenase
  • Protein Kinases
  • Alprostadil
  • Adenosine
  • 1-Methyl-3-isobutylxanthine
Topics
  • 1-Methyl-3-isobutylxanthine (pharmacology)
  • 2-Chloroadenosine
  • Adenosine (analogs & derivatives, pharmacology)
  • Alprostadil (pharmacology)
  • Animals
  • Brain (metabolism)
  • Calcimycin (pharmacology)
  • Colforsin (pharmacology)
  • Cricetinae
  • Cyclic AMP (metabolism, pharmacology)
  • Electrophoresis, Polyacrylamide Gel
  • Embryo, Mammalian
  • Enkephalin, Leucine (analogs & derivatives, pharmacology)
  • Enkephalin, Leucine-2-Alanine
  • Hybrid Cells (metabolism)
  • Immunoassay
  • Mice
  • Nerve Tissue Proteins (metabolism)
  • Neuroblastoma (metabolism)
  • Neurotransmitter Agents (pharmacology)
  • Phosphoproteins (metabolism)
  • Phosphorylation
  • Protein Kinases (metabolism)
  • Serotonin (pharmacology)
  • Tumor Cells, Cultured
  • Tyrosine 3-Monooxygenase (metabolism)

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