Chao Wun S, Serpe Marcelo D, Jia Ying, Shelver Weilin L, Anderson James V, Umeda Masaaki
USDA-Agricultural Research Service, Biosciences Research Laboratory, 1605 Albrecht Blvd., Fargo, ND 58105-5674, USA.
Plant Mol Biol. 2007 Feb;63(3):365-79. doi: 10.1007/s11103-006-9094-y. Epub 2006 Oct 25.
Leafy spurge (Euphorbia esula L.) is a deep-rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (Accession No. AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell-cycle progression was identified, and its function was confirmed based on its ability to rescue a yeast temperature-sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF;1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell-cycle signaling pathways.
乳浆大戟（Euphorbia esula L.）是一种深根多年生杂草，通过种子以及位于冠部和根部的地下不定芽进行繁殖。为了加深我们对种子萌发和营养繁殖过程中生长发育的理解，我们鉴定了一个乳浆大戟基因（登录号AF230740），该基因编码一种参与细胞周期进程的细胞周期蛋白依赖性激酶激活激酶（Ee;CDKF;1），并基于其拯救酵母温度敏感型CAK突变体（GF2351）的能力以及体外激酶测定，证实了其功能。Ee;CDKF;1的定点诱变表明，两个苏氨酸残基（Thr291和Thr296）相互负责分子内自磷酸化以及磷酸化其底物蛋白细胞周期蛋白依赖性激酶（CDK）。针对Ee;CDKF;1蛋白或磷酸化的Ee;CDKF;1肽段[NERYGSL(pT)SC]产生的多克隆抗体，用于检测种子萌发和芽生长过程中CDKF;1的丰度和磷酸化情况。干燥或吸胀种子中CDKF;1的水平低于萌发种子或幼苗。在不定芽发育过程中也观察到了CDKF;1的差异；小芽中的CDKF;1水平似乎高于大芽。使用CDKF;1蛋白或磷酸化肽段制备的多克隆抗体均检测到了相似的CDKF;1表达模式。这些结果表明，Thr291在体内持续磷酸化，并与Ee;CDKF;1活性相关。我们的结果进一步表明，大多数组织中维持着一定水平的CDKF;1活性，这对于调节细胞周期信号通路早期步骤的酶来说可能是一个重要现象。
