Kiriyama H, Nanmori T, Hari K, Matsuoka D, Fukami Y, Kikkawa U, Yasuda T
Graduate School of Science and Technology, Division of Molecular Science, Kobe University, Japan.
FEBS Lett. 1999 Apr 30;450(1-2):95-100. doi: 10.1016/s0014-5793(99)00472-x.
A gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP-dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4-kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA-selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms.
从光合鞭毛虫纤细裸藻（Euglena gracilis Z）中鉴定出一个名为epk2的基因，该基因编码一种蛋白激酶的氨基酸序列。同源性搜索和系统发育分析表明，epk2推导的氨基酸序列与环磷酸腺苷依赖性蛋白激酶（PKA）催化亚基的序列最为相似。Northern印迹分析显示，裸藻细胞表达该基因的1.4kb转录本。当EPK2蛋白与PKA的大鼠调节亚基在培养的哺乳动物细胞中共表达时，这两种蛋白会被共免疫沉淀。在与环磷酸腺苷孵育的细胞裂解物中未检测到EPK2与PKA的大鼠调节亚基的结合。从转染细胞中免疫沉淀的EPK2使PKA的合成肽底物肯普肽（Kemptide）磷酸化，并且该磷酸化被PKA选择性蛋白激酶抑制剂PKI抑制。这些结果表明，EPK2是光合鞭毛虫中的一种PKA同源物，这是光合生物中存在PKA催化亚基的首个证据。
