Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation
Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation
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DOI：
10.1074/jbc.m307151200
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发表时间：
2003-10-17
影响因子：
4.8
通讯作者：
Randall, SK
中科院分区：
文献类型：
--
作者：
Alsheikh, MK;Heyen, BJ;Randall, SK
ERD14 蛋白（脱水早期反应）是脱水蛋白家族的一员，它会因脱水相关的环境压力而积累。在这里，我们展示了拟南芥脱水蛋白 ERD14 具有离子结合特性。 ERD14 是酪蛋白激酶 II 的体外底物；磷酸化导致 SDS-PAGE 凝胶上表观分子量的变化和钙结合活性的增加。磷酸化蛋白结合的钙明显多于非磷酸化蛋白，解离常数为 120 μM，每 mol 蛋白结合 2.86 mol 钙。 ERD14 被冷处理组织提取物磷酸化，表明该蛋白的磷酸化状态可能受到冷调节激酶或磷酸酶的调节。从拟南芥提取物中纯化的 ERD14 的钙结合特性与磷酸化大肠杆菌表达的 ERD14 相当。每摩尔 ERD14 掺入约 2 摩尔磷酸盐，表明至少有两个磷酸化位点。蛋白质印迹分析证实苏氨酸和丝氨酸是 ERD14 上可能的磷酸化位点。利用基质辅助激光解吸电离飞行时间/质谱法，我们鉴定了体内和体外磷酸化 ERD14 中都存在的五种磷酸化肽。我们的结果表明，多聚丝氨酸 (S) 结构域很可能是 ERD14 中负责钙结合激活的磷酸化位点。
The ERD14 protein ( early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 muM and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/ mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine ( S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.