Szczegielniak Jadwiga, Klimecka Maria, Liwosz Aneta, Ciesielski Arkadiusz, Kaczanowski Szymon, Dobrowolska Grazyna, Harmon Alice C, Muszyńska Grazyna
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Plant Physiol. 2005 Dec;139(4):1970-83. doi: 10.1104/pp.105.066472. Epub 2005 Nov 18.
Using protein sequence data obtained from a calcium- and phospholipid-regulated protein kinase purified from maize (Zea mays), we isolated a cDNA encoding a calcium-dependent protein kinase (CDPK), which we designated ZmCPK11. The deduced amino acid sequence of ZmCPK11 includes the sequences of all the peptides obtained from the native protein. The ZmCPK11 sequence contains the kinase, autoregulatory, and calmodulin-like domains typical of CDPKs. Transcripts for ZmCPK11 were present in every tested organ of the plant, relatively high in seeds and seedlings and lower in stems, roots, and leaves. In leaves, kinase activity and ZmCPK11 mRNA accumulation were stimulated by wounding. The level of ZmCPK11 is also increased in noninjured neighboring leaves. The results suggest that the maize protein kinase is involved in a systemic response to wounding. Bacterially expressed glutathione S-transferase (GST)-ZmCPK11 was catalytically active in a calcium-dependent manner. Like the native enzyme, GST-ZmCPK11 was able to phosphorylate histone III-S and Syntide 2. Phosphorylation of histone was stimulated by phosphatidylserine, phosphatidylinositol, and phosphatidic acid, whereas phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, diolein, and cardiolipin did not increase the enzymatic activity. Autophosphorylation of GST-ZmCPK11 was stimulated by calcium and by phosphatidic acid and, to a lesser extent, by phosphatidylserine. Phosphatidylcholine did not affect autophosphorylation. These data unequivocally identify the maize phospholipid- and calcium-regulated protein kinase, which has protein kinase C-like activity, as a CDPK, and emphasize the potential that other CDPKs are regulated by phospholipids in addition to calcium.
利用从玉米（Zea mays）中纯化得到的一种钙和磷脂调节蛋白激酶获得的蛋白质序列数据，我们分离出了一个编码钙依赖性蛋白激酶（CDPK）的cDNA，我们将其命名为ZmCPK11。ZmCPK11推导的氨基酸序列包含了从天然蛋白中获得的所有肽段的序列。ZmCPK11序列包含了CDPK典型的激酶、自身调节和类钙调蛋白结构域。ZmCPK11的转录本存在于植物的每个测试器官中，在种子和幼苗中相对较高，而在茎、根和叶中较低。在叶片中，激酶活性和ZmCPK11 mRNA积累受到创伤的刺激。未受伤的相邻叶片中ZmCPK11的水平也会升高。结果表明，玉米蛋白激酶参与了对创伤的系统性反应。细菌表达的谷胱甘肽S-转移酶（GST）-ZmCPK11以钙依赖性方式具有催化活性。与天然酶一样，GST-ZmCPK11能够磷酸化组蛋白III-S和合成肽2。磷脂酰丝氨酸、磷脂酰肌醇和磷脂酸刺激组蛋白的磷酸化，而磷脂酰胆碱、溶血磷脂酰胆碱、磷脂酰乙醇胺、二油精和心磷脂不会增加酶活性。GST-ZmCPK11的自身磷酸化受到钙和磷脂酸的刺激，在较小程度上也受到磷脂酰丝氨酸的刺激。磷脂酰胆碱不影响自身磷酸化。这些数据明确地将具有蛋白激酶C样活性的玉米磷脂和钙调节蛋白激酶鉴定为一种CDPK，并强调了其他CDPK除了受钙调节外还可能受磷脂调节的可能性。
