A cGMP-dependent protein kinase is implicated in wild-type motility in C. elegans.
A cGMP-dependent protein kinase is implicated in wild-type motility in C. elegans.
复制标题
cGMP 依赖性蛋白激酶与线虫的野生型运动有关。
DOI：
10.1046/j.1471-4159.2001.00131.x
复制
发表时间：
2001
影响因子：
4.7
通讯作者：
Uhler,MD
中科院分区：
文献类型：
--
作者：
Stansberry,J;Baude,EJ;Taylor,MK;Chen,PJ;Jin,SW;Ellis,RE;Uhler,MD
在哺乳动物中，环GMP和cGMP依赖性蛋白激酶（cGK）参与了许多神经元功能的调节，包括突触功效的长时程增强和长时程抑制。为了建立一个研究cGK神经元功能的模型系统，我们克隆并鉴定了gk-1基因。一种方法的组合表明，cgk-1产生三种转录本，它们的第一个外显子不同，但长度相似。北方分析表明，C.用设计用于与所有三种转录物杂交的探针进行elegansRNA，证实了在发育的所有阶段都存在主要的3.0 kbcgk-1转录物。 为了确定CGK-1C蛋白是否是cGMP依赖性蛋白激酶，在Sf 9细胞中表达CGK-1C并纯化。CGK-1C对cGMP和cAMP的Ka分别为190 ± 14 nm和18.4 ± 2 µ m。 此外，CGK-1C以cGMP依赖性方式进行自磷酸化，并被常用的cGK抑制剂KT 5823抑制。为了确定哪些细胞表达CGK-1C，使用来自CGK-1C启动子的2.4 kb DNA片段来驱动GFP表达。CGK‐1C报告基因构建体在腹神经索和其他几种神经元以及咽和肠的边缘细胞中强烈表达。最后，RNA介导的CGK-1干扰导致线虫幼虫的运动缺陷。这些结果首次证明了cGMP依赖性蛋白激酶存在于秀丽隐杆线虫的神经元中，并表明该激酶是正常运动所必需的。
In mammals, cyclic GMP and cGMP‐dependent protein kinases (cGKs) have been implicated in the regulation of many neuronal functions including long‐term potentiation and long‐term depression of synaptic efficacy. To developCaenorhabditis elegansas a model system for studying the neuronal function of the cGKs, we cloned and characterized thecgk‐1gene. A combination of approaches showed thatcgk‐1produces three transcripts, which differ in their first exon but are similar in length. Northern analysis ofC. elegansRNA, performed with a probe designed to hybridize to all three transcripts, confirmed that a major 3.0 kbcgk‐1transcript is present at all stages of development. To determine if the CGK‐1C protein was a cGMP‐dependent protein kinase, CGK‐1C was expressed inSf9 cells and purified. CGK‐1C shows a Kaof 190 ± 14 nmfor cGMP and 18.4 ± 2 µmfor cAMP. Furthermore, CGK‐1C undergoes autophosphorylation in a cGMP‐dependent manner and is inhibited by the commonly used cGK inhibitor, KT5823. To determine which cells expressed CGK‐1C, a 2.4‐kb DNA fragment from the promoter of CGK‐1C was used to drive GFP expression. The CGK‐1C reporter construct is strongly expressed in the ventral nerve cord and in several other neurons as well as the marginal cells of the pharynx and intestine. Finally, RNA‐mediated interference of CGK‐1 resulted in movement defects in nematode larvae. These results provide the first demonstration that cGMP‐dependent protein kinase is present in neurons ofC.elegansand show that this kinase is required for normal motility.