Townsend R R, Lipniunas P H, Tulk B M, Verkman A S
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Protein Sci. 1996 Sep;5(9):1865-73. doi: 10.1002/pro.5560050912.
HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.
采用高效液相色谱-电喷雾质谱法来鉴定细菌表达的囊性纤维化跨膜传导调节因子（CFTR）片段上的磷酸化位点，该片段包含第一个核苷酸结合结构域（NBD1）和调节结构域（R）。对NBD1-R（CFTR第404-830位残基）进行胰蛋白酶消化后，分析蛋白激酶A（PKA）处理后的所有可能肽段和磷酸化肽段（共118种），这些肽段和磷酸化肽段在“高概率”PKA共有序列（R-R/K-X-S/T、R-X-X-S/T和R-X-S/T）中含有丝氨酸残基。使用三个标准来确定磷酸化位点：（1）胰蛋白酶肽段预测平均分子量增加80道尔顿；（2）与阶梯能量碰撞诱导的PO3-离子共洗脱；（3）磷酸化肽段和未修饰肽段的相对洗脱位置。NBD1和R结构域中八个双碱性位点（第422、660、700、712、737、768、795和813位）的丝氨酸残基发生了磷酸化，这一模式与全长CFTR中观察到的相似。CFTR中在体内发生磷酸化的第753位丝氨酸未被磷酸化。其余潜在的PKA位点，即丝氨酸489、丝氨酸519、丝氨酸557、丝氨酸670和苏氨酸788，未被磷酸化。“低概率”PKA位点（不包含精氨酸残基的位点）未被磷酸化。结果表明，CFTR的分离结构域为研究CFTR内磷酸化的生化和结构效应建立了有用的模型。本研究中的质谱方法应有助于确定CFTR在体外和体内的磷酸化位点。
