Lytle C, Forbush B
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1992 Dec 15;267(35):25438-43.
We determined the relationship between the activation state and phosphorylation state of the Na-K-Cl cotransport protein in tubules isolated from the shark rectal gland, a prototypic chloride-secreting epithelium. In response to cAMP-dependent secretagogues (e.g. vasoactive intestinal peptide, adenosine, and forskolin) or osmotically induced changes in cell volume, the activation state of the cotransport protein (assessed from measurements of loop diuretic binding) increased 5-10 fold. The response was temporally associated with a comparable increase (3-9 fold) in cotransport protein phosphorylation. Graded changes in cotransporter activation evoked proportional changes in cotransporter phosphorylation. Under the conditions of our experiments, the 195-kDa cotransporter was the only membrane protein whose phosphorylation state increased conspicuously in response to both cAMP and cell shrinkage. Both stimuli promoted phosphorylation of the cotransport protein at serine and threonine residues. One of the cAMP-sensitive phosphoacceptors was found within a segment of the cotransport protein comprised of a sequence (Phe-Gly-His-Asn-Thr*-Ile-Asp-Ala-Val-Pro) that corresponds to a segment of the Na-K-Cl cotransport protein predicted by cDNA analysis, where the phosphoacceptor (Thr*) is threonine 189. Incubation of rectal gland tubules with K-252a or H-8, structurally different protein kinase inhibitors, rendered the cotransporter insensitive to both cAMP and cell shrinkage. We conclude that the rectal gland Na-K-Cl cotransport protein is regulated by direct reversible phosphorylation at serine and threonine sites.
我们确定了从鲨鱼直肠腺（一种典型的氯化物分泌上皮）分离出的肾小管中钠 - 钾 - 氯共转运蛋白的激活状态与磷酸化状态之间的关系。响应于cAMP依赖性促分泌剂（如血管活性肠肽、腺苷和福斯可林）或渗透压诱导的细胞体积变化，共转运蛋白的激活状态（通过襻利尿剂结合测量评估）增加了5至10倍。该反应在时间上与共转运蛋白磷酸化的类似增加（3至9倍）相关。共转运体激活的分级变化引起共转运体磷酸化的成比例变化。在我们的实验条件下，195 kDa的共转运体是唯一一种其磷酸化状态在响应cAMP和细胞收缩时显著增加的膜蛋白。两种刺激均促进共转运蛋白在丝氨酸和苏氨酸残基处的磷酸化。发现cAMP敏感的磷酸接受体之一位于共转运蛋白的一段序列（Phe - Gly - His - Asn - Thr* - Ile - Asp - Ala - Val - Pro）中，该序列对应于cDNA分析预测的钠 - 钾 - 氯共转运蛋白的一段，其中磷酸接受体（Thr*）是苏氨酸189。用结构不同的蛋白激酶抑制剂K - 252a或H - 8孵育直肠腺小管，使共转运体对cAMP和细胞收缩均不敏感。我们得出结论，直肠腺钠 - 钾 - 氯共转运蛋白受丝氨酸和苏氨酸位点的直接可逆磷酸化调节。
