Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation
Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation
复制标题
DOI：
10.1105/tpc.10.3.451
复制
发表时间：
1998-03-01
期刊：
影响因子：
11.6
通讯作者：
Kjellbom, P
中科院分区：
文献类型：
--
作者：
Johansson, I;Karlsson, M;Kjellbom, P
PM28 A是菠菜叶片质膜的主要内在蛋白和主要磷蛋白，PM28 A的磷酸化在体内依赖于质外体水势，在体外依赖于亚微摩尔浓度的Ca ~（2+）。野生型和突变体形式的PM28 A，其中假定的磷酸化位点已被敲除，表达在非洲爪蟾卵母细胞，并在存在或不存在的蛋白激酶（K252 a）或蛋白磷酸酶（冈田酸）的抑制剂的抑制剂的存在下，测定由此产生的渗透水渗透性的增加。结果表明，PM28 A的水通道活性受两个丝氨酸残基的磷酸化调节，第一胞质环中的Ser-115和C-末端区域中的Ser-274。用P-32-正磷酸标记菠菜叶并随后对PM 28 A衍生肽进行测序，证明Ser-274在体内被磷酸化，而Ser-115（在所有植物质膜水通道蛋白中保守的残基）的磷酸化不能被证明，这表明，PM28 A的274个氨基酸残基在体内响应于增加的质外体水势而被磷酸化，并且在体内被去磷酸化。对降低水势的反应。综上所述，我们的研究结果表明，PM28 A在维持细胞水平衡方面发挥着积极作用。
PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein, Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+, Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with P-32-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated, This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance.