Phosphorylation of the nicotinic acetylcholine receptor in myotube-cholinergic neuron cocultures.
Acetylcholine receptor (AChR) stability in the postsynaptic membrane is affected by serine kinases. AChR are phosphorylated by protein kinase C (PKC) and PKA, and we have shown that activation of PKA and PKC have opposite effects on AChR stability and that this may play some role in the selective, activity-dependent synapse loss that occurs during development of the neuromuscular junction. Myotube cultures with and without added spinal motor neurons were probed with immunoaffinity-purified antibodies prepared against phosphorylated peptides with amino acid sequences from different AChR subunits. Different treatments activating PKC (phorbol 12-myristate 13-acetate; PMA) or PKA (dibutyryl cyclic adenosine monophosphate; cAMP) or blocking electrical activity (tetrodotoxin; TTX) of the cocultures were chosen because of their known effects, direct or indirect, on receptor stability. We asked whether the phospho-specific antibody staining in conjunction with alpha-bungarotoxin (BTX) identification of AChR aggregates could provide a direct demonstration of changes in receptor phosphorylation produced by the treatments. We found that PMA treatment did increase phosphorylation of the delta subunit and cAMP increased phosphorylation of the epsilon subunit relative to total BTX labeling in muscle-nerve cocultures, but not in muscle-only cultures. Blockade of electrical activity with TTX increased the incidence of aggregates that showed no phospho-epsilon staining. Myotube cultures grown in the absence of neurons did not show the responses of myotubes in cocultures. The results show that manipulations that alter receptor stability also produce changes in receptor phosphorylation. We suggest that phosphorylation may be a mechanism mediating the changes in receptor stability.
Lanuza
Maria A
MA
Unitat d'Histologia i Neurobiologia (UHN), Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain. mariaangel.lanuza@urv.net
Gizaw
Rahel
R
Viloria
Alexa
A
González
Carmen M
CM
Besalduch
Núria
N
Dunlap
Veronica
V
Tomàs
Josep
J
Nelson
Phillip G
PG
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002038:Bungarotoxins / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D007150:Immunohistochemistry; D051379:Mice; D009046:Motor Neurons / Q000166:cytology / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000196:embryology* / Q000254:growth & development / Q000294:innervation*; D009469:Neuromuscular Junction / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D021122:Protein Subunits / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002038:Bungarotoxins; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D026941:Sodium Channel Blockers; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate