Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant
Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant
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DOI：
10.1104/pp.103.035238
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发表时间：
2004-07-01
期刊：
影响因子：
7.4
通讯作者：
Cushman, JC
中科院分区：
文献类型：
--
作者：
Chehab, EW;Patharkar, OR;Cushman, JC
从普通冰草中分离到一种盐和脱水胁迫响应性钙依赖蛋白激酶（CDPK）。McCPK 1在体外经历肉豆蔻酰化，但不经历棕榈酰化。去除N-末端肉豆蔻酸受体位点部分降低McCPK 1质膜（PM）定位确定的瞬时表达的绿色荧光蛋白融合在微粒轰击细胞。N-末端结构域（氨基酸1-70）的去除完全废除PM本地化，这表明豆蔻酰化和可能的N-末端结构域有助于膜协会的激酶。重组，大肠杆菌表达，全长McCPK 1蛋白是催化活性的钙依赖性的方式（K-0.5 = 0.15妈妈）。重组McCPK 1的自磷酸化在体外观察到至少两个不同的Ser残基，与两个位点的位置被映射到Ser-62和Ser-420。在Ser-62或Ser-420自磷酸化位点的Ala取代导致相对于野生型McCPK 1对组蛋白H1底物的激酶活性略有增加。相比之下，在这两个网站的Ala取代导致激酶活性急剧下降，相对于野生型McCPK 1使用组蛋白H1作为底物。McCPK 1经历了一个可逆的变化，在亚细胞定位从PM到细胞核，内质网，肌动蛋白微丝的细胞骨架，在响应湿度的减少，确定由瞬时表达的McCPK 1-绿色荧光蛋白融合在microprojectile-bombarded细胞，并证实了亚细胞分级分离和蛋白质印迹分析6 × His标记的McCPK 1。
A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K-0.5 = 0.15 mum). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.