Tanimura A, Kurihara K, Reshkin S J, Turner R J
Clinical Investigations and Patient Care Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Oct 20;270(42):25252-8. doi: 10.1074/jbc.270.42.25252.
We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na(+)-K(+)-2Cl- cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na(+)-K(+)-2Cl- cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the beta-adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (approximately 20 nM) was in excellent agreement with its previously demonstrated up-regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. AlF(-)4 treatment, which results in an up-regulation of cotransport activity comparable with that observed with isoproterenol (approximately 6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (approximately 3-fold and approximately 6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca(2+)-mobilizing agent thapsigargin, which is able to fully mimic the up-regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na(+)-K(+)-2Cl- cotransporter.
我们在大鼠腮腺腺泡中鉴定出一种175-kDa的膜磷蛋白（pp175），其特性与先前在该组织中通过功能和生化方法鉴定的Na(+)-K(+)-2Cl-协同转运蛋白密切相关。pp175是唯一能被抗Na(+)-K(+)-2Cl-协同转运蛋白抗体免疫沉淀的磷蛋白，也是腺泡短暂（45秒）暴露于β-肾上腺素能激动剂异丙肾上腺素后，其磷酸化状态发生显著改变的唯一膜蛋白。磷酸肽图谱分析为pp175上的三个磷酸化位点提供了证据，其中只有一个位点在异丙肾上腺素处理后被标记。异丙肾上腺素对pp175磷酸化的半数最大效应（约20 nM）与其先前证明的对协同转运活性的上调作用高度一致。在用渗透性cAMP类似物和福斯可林处理腺泡后，也观察到pp175磷酸化增加，这些条件同样已被证明可上调协同转运蛋白。结合我们实验室早期的结果，这些数据提供了有力证据，表明这些试剂对协同转运蛋白的上调是由于蛋白激酶A介导的直接磷酸化。AlF(-)4处理导致协同转运活性上调，与异丙肾上腺素观察到的上调程度相当（约6倍），引起pp175磷酸化类似增加。然而，高渗收缩和用蛋白磷酸酶抑制剂花萼海绵诱癌素A处理，这两种处理也上调协同转运蛋白（分别约3倍和约6倍），但并未导致磷酸化水平改变。此外，尽管用毒蕈碱激动剂卡巴胆碱处理腺泡会导致协同转运活性显著上调以及pp175伴随磷酸化，但用钙动员剂毒胡萝卜素处理时，未见pp175磷酸化，而毒胡萝卜素能够完全模拟卡巴胆碱对转运活性的上调作用。综上所述，这些结果表明直接磷酸化只是促分泌剂诱导大鼠腮腺Na(+)-K(+)-2Cl-协同转运蛋白调节所涉及的机制之一。
