Lee J Y, Yoo B C, Harmon A C
Program in Plant Molecular and Cellular Biology, Department of Botany, University of Florida, Gainesville 32611-8526, USA.
Biochemistry. 1998 May 12;37(19):6801-9. doi: 10.1021/bi980062q.
Calmodulin-like domain protein kinases (CDPKs) are a family of calcium- but not calmodulin-dependent protein kinases found in a wide variety of plants and in protists. CDPKs are encoded by large multigene families, and to assess whether family members play distinct or redundant roles in vivo, we characterized soybean CDPK isoforms alpha, beta, and gamma, which share 60-80% identity in amino acid sequence. RNA blot analysis showed that the three CDPKs were expressed in most plant tissues examined and in suspension-cultured soybean cells. Recombinant CDPKalpha, -beta, and -gamma phosphorylated peptide substrates containing the four-residue motif R/K-X-X-S/T, but CDPKalpha was the most selective for residues outside of the motif. The CDPKs were inhibited by the general protein kinase inhibitors K252a and staurosporine and by calphostin C, which is an inhibitor of protein kinase C. The calcium-binding properties of each CDPK were distinct. The Kd's for Ca2+ determined by flow dialysis in the absence of substrates were 51, 1.4, and 1.6 micro M for CDPKalpha, -beta, and -gamma, respectively. In the presence of the peptide substrate syntide-2 the Kd of CDPKalpha decreased to 0.6 microM. Also, the sensitivity of this isoenzyme's activity to calcium varied with protein substrate. The concentrations of Ca2+ required for half-maximal activity (K0.5) for each CDPK with syntide-2 as substrate were 0.06, 0.4, and 1 micro M, respectively. These results show that members of the CDPK family differ in biochemical properties and support the hypothesis that each isoform may have a distinct role in calcium signal transduction.
类钙调蛋白结构域蛋白激酶（CDPKs）是一类在多种植物和原生生物中发现的依赖钙而非钙调蛋白的蛋白激酶家族。CDPKs由大型多基因家族编码，为了评估家族成员在体内是否发挥不同或冗余的作用，我们对大豆CDPK亚型α、β和γ进行了表征，它们在氨基酸序列上具有60 - 80%的同一性。RNA印迹分析表明，这三种CDPK在大多数检测的植物组织以及悬浮培养的大豆细胞中均有表达。重组CDPKα、-β和-γ可磷酸化含有四残基基序R/K-X-X-S/T的肽底物，但CDPKα对该基序之外的残基具有最高的选择性。这些CDPK被通用蛋白激酶抑制剂K252a和星形孢菌素以及蛋白激酶C抑制剂钙磷蛋白C所抑制。每种CDPK的钙结合特性各不相同。在无底物情况下通过流动透析测定的CDPKα、-β和-γ对Ca2+的解离常数（Kd）分别为51、1.4和1.6 μM。在存在肽底物syntide-2时，CDPKα的Kd降至0.6 μM。此外，这种同工酶的活性对钙的敏感性随蛋白质底物而变化。以syntide-2为底物时，每种CDPK达到最大活性一半时所需的Ca2+浓度（K0.5）分别为0.06、0.4和1 μM。这些结果表明，CDPK家族成员在生化特性上存在差异，并支持了每种亚型可能在钙信号转导中具有不同作用的假说。
