Lytle C
Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521, USA.
J Biol Chem. 1997 Jun 13;272(24):15069-77. doi: 10.1074/jbc.272.24.15069.
Na-K-Cl cotransport activity in duck erythrocytes increases approximately 10-fold in response to osmotic cell shrinkage, norepinephrine, fluoride, or calyculin-A (an inhibitor of type-1 and -2a phosphatases). To assess whether all four stimuli promote phosphorylation of the cotransport protein and whether this phosphorylation is catalyzed by the same kinase, the cotransporter was isolated from erythrocytes by immunoprecipitation and its pattern of phosphorylation was evaluated. Each stimulus evoked proportionate increases in cotransporter activity and phosphorylation. No two stimuli in combination evoked greater activation and phosphorylation than did the more potent of the two stimuli acting alone. Phosphoamino acid analysis of the cotransport protein indicated that phosphorylation occurs at serine and threonine residues. Phosphopeptide mapping revealed a distinctive pattern of 8 major tryptic phosphopeptides, none of which were significantly phosphorylated in the unstimulated state. Maps of cotransporters activated by the four different stimuli were indistinguishable. Measurements of phosphorylation stoichiometry indicated that each cotransporter acquires approximately 5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. Staurosporine, a kinase inhibitor with broad selectivity, inhibited each stimulus equipotently (IC50 approximately 0.7 microM). Staurosporine promptly reversed cotransporter activity and phosphorylation when added to shrinkage-stimulated but not to calyculin-stimulated cells, indicating that it enters the cell rapidly and blocks phosphorylation. These results suggest that cell shrinkage, cAMP, fluoride, and calyculin-A promote the phosphorylation of the Na-K-Cl cotransport protein at a similar constellation of serine and threonine residues. It is proposed that all modes of stimulation ultimately involve the same protein kinase.
鸭红细胞中的钠 - 钾 - 氯协同转运活性在细胞因渗透作用而皱缩、受到去甲肾上腺素、氟化物或花萼海绵诱癌素A（一种1型和2a型磷酸酶抑制剂）刺激时会增加约10倍。为了评估这四种刺激是否都能促进协同转运蛋白的磷酸化，以及这种磷酸化是否由同一种激酶催化，通过免疫沉淀从红细胞中分离出协同转运蛋白，并对其磷酸化模式进行评估。每种刺激均引起协同转运蛋白活性和磷酸化的相应增加。没有两种刺激联合作用能比单独作用更强的那种刺激引发更大的激活和磷酸化。对协同转运蛋白的磷酸氨基酸分析表明，磷酸化发生在丝氨酸和苏氨酸残基上。磷酸肽图谱显示出8种主要胰蛋白酶磷酸肽的独特模式，在未受刺激状态下这些磷酸肽均未发生显著磷酸化。由四种不同刺激激活的协同转运蛋白图谱无法区分。磷酸化化学计量的测量表明，每个协同转运蛋白从肿胀细胞中的无活性状态转变为皱缩细胞中的活性状态时会获得约5个磷酸基团。星形孢菌素是一种具有广泛选择性的激酶抑制剂，能同等程度地抑制每种刺激（半数抑制浓度约为0.7微摩尔）。当添加到经皱缩刺激而非花萼海绵诱癌素刺激的细胞中时，星形孢菌素能迅速逆转协同转运蛋白的活性和磷酸化，这表明它能快速进入细胞并阻断磷酸化。这些结果表明，细胞皱缩、环磷酸腺苷、氟化物和花萼海绵诱癌素A在丝氨酸和苏氨酸残基的相似位点促进钠 - 钾 - 氯协同转运蛋白的磷酸化。有人提出，所有刺激模式最终都涉及同一种蛋白激酶。
