Huganir R L, Miles K, Greengard P
Proc Natl Acad Sci U S A. 1984 Nov;81(22):6968-72. doi: 10.1073/pnas.81.22.6968.
Postsynaptic membranes from the electric organ of Torpedo californica, rich in the nicotinic acetylcholine receptor, were shown to contain an endogenous tyrosine protein kinase. This endogenous kinase phosphorylated three major proteins with molecular masses corresponding to 50 kDa, 60 kDa, and 65 kDa. The phosphorylation of these three proteins occurred exclusively on tyrosine residues under the experimental conditions used and was abolished by 0.1% Nonidet P-40 and stimulated by Mn2+. The 50-kDa, and 60-kDa, and 65-kDa phosphoproteins were demonstrated to be the beta, gamma, and delta subunits, respectively, of the nicotinic acetylcholine receptor by purification of the phosphorylated receptor using affinity chromatography. The endogenous tyrosine kinase specifically phosphorylated the beta, gamma, and delta subunits rapidly to a final stoichiometry of approximately equal to 0.5 mol of phosphate per mol of sub-unit. Two-dimensional phosphopeptide mapping of the phosphorylated beta, gamma, and delta subunits, after limit proteolysis with trypsin or thermolysin, indicated that each subunit was phosphorylated on a single site. Locations are proposed for the amino acid residues phosphorylated on the receptor by the tyrosine-specific protein kinase and by two other protein kinases (cAMP-dependent protein kinase and protein kinase C) which phosphorylate the receptor.
电鳐电器官的突触后膜富含烟碱型乙酰胆碱受体，已证明其含有一种内源性酪氨酸蛋白激酶。这种内源性激酶使三种主要蛋白质磷酸化，其分子量分别对应于50 kDa、60 kDa和65 kDa。在所用实验条件下，这三种蛋白质的磷酸化仅发生在酪氨酸残基上，0.1%的诺乃洗涤剂P-40可消除这种磷酸化，而Mn2+可刺激其发生。通过使用亲和色谱法纯化磷酸化受体，证明50 kDa、60 kDa和65 kDa的磷蛋白分别是烟碱型乙酰胆碱受体的β、γ和δ亚基。内源性酪氨酸激酶特异性地使β、γ和δ亚基快速磷酸化，最终化学计量比约为每摩尔亚基0.5摩尔磷酸盐。在用胰蛋白酶或嗜热菌蛋白酶进行有限度蛋白水解后，对磷酸化的β、γ和δ亚基进行二维磷酸肽图谱分析，表明每个亚基在单个位点被磷酸化。本文提出了酪氨酸特异性蛋白激酶以及另外两种使该受体磷酸化的蛋白激酶（环磷酸腺苷依赖性蛋白激酶和蛋白激酶C）在受体上磷酸化的氨基酸残基的位置。
