Phosphorylation of membrane-associated proteins by phorbol esters in isolated bag cell neurons of Aplysia.
Following brief synaptic stimulation, the bag cell neurons in the abdominal ganglion of Aplysia undergo a series of changes in electrophysiological and secretory properties that triggers egg laying behavior. Activation of protein kinase C appears to play an important role in these changes and, in particular, causes the unmasking of a new species of voltage-dependent calcium channel. We have now used isolated bag cell neurons maintained in cell culture to study changes in protein phosphorylation that are induced by exposure to an activator of protein kinase C. Primary cultures of bag cell neurons were labeled with 32P orthophosphate and then incubated with either tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, or with an inactive phorbol ester. When protein extracts were separated with 2D electrophoresis approximately 100 phosphoproteins could be distinguished. Only four of these proteins, with molecular weights of 20, 32, 200, and 250 kD, underwent a reproducible increase in the extent of phosphorylation of at least twofold in response to TPA. TPA-induced changes in phosphate incorporation were blocked by pretreatment with the protein kinase C inhibitor H7. One of the TPA-regulated phosphoproteins was localized in a plasma membrane-containing fraction and was sensitive to trypsin treatment of intact cells, suggesting that it is a membrane protein with sites exposed to the extracellular medium. Two of the other TPA-regulated phosphoproteins may be associated with the inner face of the plasma membrane. Our results indicate that only a small number of proteins undergo a major change in phosphorylation state following the activation of protein kinase C in isolated bag cell neurons. One or more of these proteins may contribute to the unmasking of the calcium channels.
Azhderian
E M
EM
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Kaczmarek
L K
LK
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001048:Aplysia / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D007546:Isoquinolines; D008565:Membrane Proteins; D010703:Phorbol Esters; D010879:Piperazines; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate