Yoo B C, Harmon A C
Plant Molecular and Cellular Biology Program, University of Florida, Gainesville 32611-8526, USA.
Biochemistry. 1996 Sep 17;35(37):12029-37. doi: 10.1021/bi9606612.
The activity of calmodulin-like domain protein kinase (CDPK) is regulated by the direct binding of Ca2+. Unmodified soybean CDPK alpha and a chimeric enzyme in which the calmodulin-like domain (CLD) was replaced by VU-1 calmodulin had similar values of Vmax(app) (3.19, 3.46, and 3.60, 3.93 mumol/ min/mg, respectively), and each was activated 30-70-fold by Ca2+. To determine if activation results from the binding of the CLD to the autoinhibitory (junction) domain of CDPK alpha in a manner analogous to the activation of calmodulin-dependent enzymes by calmodulin, recombinant CLD and truncation mutants of CDPK alpha were expressed in bacteria and highly purified. In blot overlays, biotinylated CLD bound to mutants containing residues 312-328 of the junction domain. In an electrophoretic mobility shift assay CLD bound synthetic peptides containing residues 318-332 in a calcium-dependent manner, providing direct evidence for binding of CLD to a site in the junction domain. Mutants of CDPK alpha from which all or part of the CLD had been deleted were constitutively inactive. Addition of 20 microM CLD to these mutants in the presence, but not the absence, of calcium stimulated their activities, but to various degrees. His6-CDPK alpha (1-328), which contained none of the CLD, was activated only 5-fold, but the activity of His6-CDPK alpha (1-398), which retained nearly half of the CLD in its sequence, was stimulated 64-fold. The latter activity approached that of unmodified CDPK alpha and was half maximal at a CLD concentration of 7 microM. Our results suggest that binding of CLD to the junction domain contributes to, but is not sufficient for activation. Although calmodulin supported full activity of the chimeric enzyme, its addition to His6-CDPK alpha (1-398) resulted in activity that was only 6% of that of the unmodified enzyme and which was half-maximal at 20 microM Arabidopsis calmodulin. These results support the conclusion that simple binding of the calmodulin-like domain to the junction domain is not sufficient for activation.
类钙调蛋白结构域蛋白激酶（CDPK）的活性受Ca2+直接结合的调节。未修饰的大豆CDPKα和一种嵌合酶（其中类钙调蛋白结构域（CLD）被VU-1钙调蛋白取代）的Vmax(app)值相似（分别为3.19、3.46和3.60、3.93 μmol/min/mg），并且每种都被Ca2+激活30 - 70倍。为了确定激活是否源于CLD以类似于钙调蛋白激活钙调蛋白依赖性酶的方式与CDPKα的自身抑制（连接）结构域结合，重组CLD和CDPKα的截短突变体在细菌中表达并高度纯化。在印迹覆盖实验中，生物素化的CLD与包含连接结构域312 - 328位残基的突变体结合。在电泳迁移率变动分析中，CLD以钙依赖性方式与包含318 - 332位残基的合成肽结合，为CLD与连接结构域中的一个位点结合提供了直接证据。已缺失全部或部分CLD的CDPKα突变体组成型无活性。在有钙但无钙时不存在的情况下，向这些突变体中添加20 μM CLD刺激了它们的活性，但程度不同。不含任何CLD的His6 - CDPKα(1 - 328)仅被激活5倍，但序列中保留了近一半CLD的His6 - CDPKα(1 - 398)的活性被刺激了64倍。后者的活性接近未修饰的CDPKα的活性，并且在CLD浓度为7 μM时达到最大活性的一半。我们的结果表明，CLD与连接结构域的结合有助于激活，但不足以激活。尽管钙调蛋白支持嵌合酶的完全活性，但将其添加到His6 - CDPKα(1 - 398)中导致的活性仅为未修饰酶活性的6%，并且在20 μM拟南芥钙调蛋白时达到最大活性的一半。这些结果支持这样的结论，即类钙调蛋白结构域与连接结构域的简单结合不足以激活。
