Muscarinic receptor- and phorbol ester-stimulated phosphorylation of protein kinase C substrates in adult and neonatal cortical slices.
The neuron-specific protein B-50 (GAP-43) is a major presynaptic substrate for protein kinase C (PKC). Phosphorylation of B-50 by PKC at serine-41 is functionally related to signal transduction in association with process outgrowth and neurotransmitter release. Thus, it is important to characterize the factors which modulate phosphorylation of B-50 by PKC. Phosphoinositide (PI)-coupled muscarinic acetylcholine receptor (mAchR) activation would be expected to increase PKC activity through production of the second messenger, diacylglycerol. To test the hypothesis that activation of mAchR also increases phosphorylation of B-50, protein phosphorylation has been examined in cerebral cortical slices in response to the cholinergic agonist, carbachol (Cch) in comparison to the phorbol ester, 4beta-phorbol 12, 13-dibutyrate (PDB), a known activator of PKC. At short times of incubation with 1 mM Cch, a concentration which maximally activates PI metabolism, increased phosphorylation of a group of synaptosomal proteins, including B-50 and myristoylated, alanine-rich C kinase substrate (MARCKS), was observed. This increase was approximately half of that obtained in response to 1 microM PDB. Differing patterns of protein phosphorylation were observed in neonatal and adult slices: neonatal samples contained more MARCKS and a PKC substrate with a Mr of 46 kDa. Phosphorylation of B-50 and MARCKS was sensitive to Cch in both cases. Immunoblotting demonstrated less m1 acetylcholine receptor (the predominant mAchR subtype coupled to PI metabolism in the cortex) in neonatal, as compared to adult, synaptosomal fractions. These results are consistent with a coupling between mAchR-stimulated PI metabolism and PKC-mediated protein phosphorylation that is developmentally regulated.
Haas
M S
MS
Departments of Biochemistry/Molecular Biology and Medicine, Medical College of Ohio, Toledo, OH 43614-5809, USA.
Dokas
L A
LA
D000367:Age Factors; D000409:Alanine; D000818:Animals; D000831:Animals, Newborn; D002217:Carbachol / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development; D004789:Enzyme Activation / Q000187:drug effects; D019922:GAP-43 Protein / Q000378:metabolism; D015151:Immunoblotting; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D009227:Myristic Acids / Q000378:metabolism; D009924:Organ Culture Techniques; D010703:Phorbol Esters / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011976:Receptors, Muscarinic / Q000502:physiology*; D013379:Substrate Specificity
D002273:Carcinogens; D019922:GAP-43 Protein; D018721:Muscarinic Agonists; D009227:Myristic Acids; D010703:Phorbol Esters; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011976:Receptors, Muscarinic; D002217:Carbachol; D011493:Protein Kinase C; D000409:Alanine