Autophosphorylation-dependent activation of a calcium-dependent protein kinase from groundnut
Autophosphorylation-dependent activation of a calcium-dependent protein kinase from groundnut
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DOI：
10.1104/pp.120.3.859
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发表时间：
1999-07-01
期刊：
影响因子：
7.4
通讯作者：
DasGupta, M
中科院分区：
文献类型：
--
作者：
Chaudhuri, S;Seal, A;DasGupta, M
钙离子依赖性蛋白激酶（CDPKs）主要存在于植物中，其一级序列中整合有钙调素样结构域。该家族的一个成员的特征来自花生（Arachis hypogea）植物，称为GnCDPK（M. DasGupta [1994] Plant Physiol 104：961-969）。GnCDPK专门使用肌球蛋白轻链合成肽（MLCpep）作为体外实验条件下的外源性底物，MLCpep是平滑肌肌球蛋白轻链（KKRPQRATSNVFS）的磷酸盐接受结构域。在这份报告中，我们表明，GnCDPK进行分子内自磷酸化。这种自身磷酸化发生在苏氨酸残基中的钙离子依赖性（K-0.5 = 0.5 μ M）和钙调素独立的方式。在饱和ATP浓度下测量时，对MLCpep的激酶活性及其对Ca 2+的敏感性不受先前自磷酸化的影响。在低ATP浓度下，重新研究了自磷酸化在外源性底物MLCpep磷酸化反应中的作用。在这种次优条件下，在MLCpep磷酸化的初始速率中观察到1至2分钟的明显滞后时间，随后活性线性增加7.5分钟。先前的自磷酸化完全取消了这个滞后期，从第1分钟看到外源性底物磷酸化的急剧上升。我们的研究结果表明，自磷酸化是激活GnCDPK的先决条件。
Ca2+-dependent protein kinases (CDPKs) containing a calmodulin-like domain integrated in their primary sequence are present primarily in plants. A member of this family was characterized from the groundnut (Arachis hypogea) plant and called GnCDPK (M. DasGupta [1994] Plant Physiol 104: 961-969). GnCDPK specifically uses the myosin light chain synthetic peptide (MLCpep), which is the phosphate-accepting domain of smooth muscle myosin light chains (KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental conditions. In this report we show that GnCDPK undergoes intramolecular autophosphorylation. This self-phosphorylation occurs in threonine residues in a Ca2+-dependent (K-0.5 = 0.5 mu M) and calmodulin-independent manner. The kinase activity toward MLCpep and its sensitivity to Ca2+ were unaffected by prior autophosphorylation when measured under saturating ATP concentrations. The role of autophosphorylation in the exogenous substrate MLCpep phosphorylation reaction was reinvestigated at low ATP concentrations. A pronounced lag time of 1 to 2 min, followed by a linear increase of activity for 7.5 min, was seen in the initial rate of MLCpep phosphorylation under such suboptimal conditions. Prior autophosphorylation completely abolished this lag phase, and a sharp rise of exogenous substrate phosphorylation was seen from the 1st min. Our results suggest that autophosphorylation is a prerequisite for the activation of GnCDPK.