Lanuza Maria A, Gizaw Rahel, Viloria Alexa, González Carmen M, Besalduch Núria, Dunlap Veronica, Tomàs Josep, Nelson Phillip G
Unitat d'Histologia i Neurobiologia (UHN), Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain.
J Neurosci Res. 2006 Jun;83(8):1407-14. doi: 10.1002/jnr.20848.
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.
突触后膜中的乙酰胆碱受体（AChR）稳定性受丝氨酸激酶影响。AChR被蛋白激酶C（PKC）和蛋白激酶A（PKA）磷酸化，并且我们已经表明PKA和PKC的激活对AChR稳定性具有相反的作用，这可能在神经肌肉接头发育过程中发生的选择性、活性依赖性突触丢失中发挥一定作用。用针对来自不同AChR亚基氨基酸序列的磷酸化肽制备的免疫亲和纯化抗体，对添加和未添加脊髓运动神经元的肌管培养物进行检测。选择不同的处理方法来激活PKC（佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯；PMA）或PKA（二丁酰环磷酸腺苷；cAMP）或阻断共培养物的电活动（河豚毒素；TTX），是因为它们对受体稳定性具有已知的直接或间接影响。我们询问磷酸化特异性抗体染色结合α - 银环蛇毒素（BTX）对AChR聚集体的鉴定，是否能直接证明处理所产生的受体磷酸化变化。我们发现，相对于肌肉 - 神经共培养物中的总BTX标记，PMA处理确实增加了δ亚基的磷酸化，而cAMP增加了ε亚基的磷酸化，但在仅含肌肉的培养物中未出现这种情况。用TTX阻断电活动增加了未显示磷酸化ε染色的聚集体的发生率。在无神经元条件下生长的肌管培养物未表现出共培养物中肌管的反应。结果表明，改变受体稳定性的操作也会导致受体磷酸化的变化。我们认为磷酸化可能是介导受体稳定性变化的一种机制。
