Nimnual A S, Chang W, Chang N S, Ross A F, Gelman M S, Prives J M
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794, USA.
Biochemistry. 1998 Oct 20;37(42):14823-32. doi: 10.1021/bi9802824.
The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.
胚胎骨骼肌细胞的神经支配表现为，肌肉表面膜上的烟碱型乙酰胆碱受体（AChR）重新分布，在神经 - 肌肉接触处形成高密度斑块。为了研究蛋白质磷酸化途径在AChR表面分布调节中的作用，我们确定了AChR δ亚基上与培养的肌管中AChR簇分散相关的磷酸化位点。我们发现，蛋白激酶C（PKC）催化的AChR磷酸化作用靶向于Ser378、Ser393和Ser450，这些位点均位于AChR δ亚基的主要胞内结构域。其中一个位点附近是蛋白激酶A（PKA）的共有靶位点（Ser377），在体外可被纯化的PKA有效磷酸化。PKC激活剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）和磷蛋白磷酸酶抑制剂冈田酸（OA）使AChR δ亚基在体外被纯化PKC磷酸化的三个丝氨酸残基上的磷酸化增加。相反，用PKA激活剂福斯可林或细胞可渗透的环磷酸腺苷类似物8 - 溴 - 环磷酸腺苷处理这些细胞，并未改变表面AChR的磷酸化状态，这表明PKA在完整的鸡肌管中不会主动磷酸化δ亚基。TPA和OA的作用包括增加在Triton X - 100中提取的表面AChR的比例，以及AChR从簇状区域扩散到肌肉细胞表面的相邻区域。这些发现表明，PKC催化的AChR δ亚基特定丝氨酸残基的磷酸化可能在这些受体的表面分布中起作用。
