Weaver Y R, Cossins A R
Department of Environmental and Evolutionary Biology, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK.
Pflugers Arch. 1996 Aug;432(4):727-34. doi: 10.1007/s004240050191.
Electroneutral salt transporters are activated and deactivated by changes to the phosphorylation status either of the transporter itself or of other, as yet unidentified, regulatory proteins. We have studied the effects of an inhibitor of protein tyrosine kinase (PTK), genistein, upon KCl cotransport in trout erythrocytes. We show that Cl-dependent K fluxes activated by physiological stimuli, i.e. oxygenation and beta-adrenergic agonists, are rapidly and completely blocked by genistein, whilst the inactive analogue of genistein, daidzein, had no effect. By contrast, the protein tyrosine phosphatase (PTP) inhibitor, vanadate (V), caused a slow but strong activation of an inactive cotransporter. This vanadate (V) activated flux was inhibited by genistein as well as by the serine/threonine phosphatase (PSP) inhibitor, calyculin A. However, genistein had no effect upon the activation of the cotransporter by the protein (serine/threonine) kinase (PSK) inhibitor, staurosporine, or by N-ethylmaleimide, which also appears to act by inhibiting a PSK. These results are consistent with a sequential scheme of at least two tyrosine phosphorylation events which lie upstream to the serine/threonine phosphorylation sites in the signal transduction pathway leading from stimulus to transporter activation. The regulation of the activity of KCl cotransporter appears to involve a complex series of phosphorylation reactions.
电中性盐转运体通过转运体自身或其他尚未确定的调节蛋白的磷酸化状态变化而被激活和失活。我们研究了蛋白酪氨酸激酶（PTK）抑制剂染料木黄酮对虹鳟红细胞中氯化钾协同转运的影响。我们发现，由生理刺激（即氧合和β-肾上腺素能激动剂）激活的氯离子依赖性钾离子通量会被染料木黄酮迅速且完全阻断，而染料木黄酮的无活性类似物大豆苷元则没有作用。相比之下，蛋白酪氨酸磷酸酶（PTP）抑制剂钒酸盐（V）会使无活性的协同转运体缓慢但强烈地激活。这种钒酸盐（V）激活的通量会被染料木黄酮以及丝氨酸/苏氨酸磷酸酶（PSP）抑制剂花萼海绵诱癌素A抑制。然而，染料木黄酮对蛋白（丝氨酸/苏氨酸）激酶（PSK）抑制剂星形孢菌素或N-乙基马来酰亚胺激活协同转运体没有影响，N-乙基马来酰亚胺似乎也是通过抑制一种PSK起作用。这些结果与至少两个酪氨酸磷酸化事件的顺序模式一致，这两个事件位于从刺激到转运体激活的信号转导途径中丝氨酸/苏氨酸磷酸化位点的上游。氯化钾协同转运体活性的调节似乎涉及一系列复杂的磷酸化反应。
