Fienberg A A, Nordstedt C, Belting H G, Czernik A J, Nairn A C, Gandy S, Greengard P, Ruddle F H
Department of Genetics, Yale University School of Medicine, Yale University, New Haven, Connecticut 06510, USA.
J Exp Zool. 1999 Apr 15;285(1):76-84.
In an effort to characterize the signal transduction mechanisms that operate to regulate homeodomain protein function, we have analyzed the phosphorylation state of two homeodomain proteins, Hoxb-6 and Hoxc-8, in vitro and in vivo. The baculovirus expression system was employed to demonstrate that Hoxb-6 is phosphorylated in Sf9 cells while Hoxc-8 is not. Using two-dimensional tryptic phosphopeptide mapping and purified protein kinases, we demonstrate that Hoxb-6 is phosphorylated in vitro by casein kinase II and cAMP-dependent protein kinase. The casein kinase II phosphorylation site was mapped to serine-214. Two-dimensional tryptic phosphopeptide mapping of immunoprecipitated Hoxb-6 from mouse embryonic spinal cords demonstrates that the same peptide phosphorylated in vitro and in Sf9 cells by casein kinase II is also phosphorylated in vivo. The conservation of this site in several homeodomain proteins from various species is discussed.
为了描述调控同源结构域蛋白功能的信号转导机制，我们在体外和体内分析了两种同源结构域蛋白Hoxb-6和Hoxc-8的磷酸化状态。利用杆状病毒表达系统证明，Hoxb-6在Sf9细胞中发生磷酸化，而Hoxc-8未发生磷酸化。通过二维胰蛋白酶磷酸肽图谱和纯化的蛋白激酶，我们证明Hoxb-6在体外被酪蛋白激酶II和cAMP依赖性蛋白激酶磷酸化。酪蛋白激酶II的磷酸化位点定位于丝氨酸-214。对从小鼠胚胎脊髓免疫沉淀的Hoxb-6进行二维胰蛋白酶磷酸肽图谱分析表明，在体外和Sf9细胞中被酪蛋白激酶II磷酸化的同一肽段在体内也发生了磷酸化。文中讨论了该位点在来自不同物种的几种同源结构域蛋白中的保守性。
