Characterization of a novel protein kinase D - Caenorhabditis elegans DKF-1 is activated by translocation-phosphorylation and regulates movement and growth in vivo
Characterization of a novel protein kinase D - Caenorhabditis elegans DKF-1 is activated by translocation-phosphorylation and regulates movement and growth in vivo
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DOI：
10.1074/jbc.m511899200
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发表时间：
2006-06-30
影响因子：
4.8
通讯作者：
Rubin, Charles S.
中科院分区：
文献类型：
--
作者：
Feng, Hui;Ren, Min;Rubin, Charles S.
蛋白激酶D （PKD）异构体是二酰基甘油（DAG）调控的信号通路中的蛋白激酶C （PKC）效应器。关键的生理过程由DAG控制，通过独特的底物特异性和pkd的细胞内分布。要理解PKD在体内稳态和信号转导中的作用，需要进一步了解PKD和PKC异构体之间的调节相互作用，分析PKC独立的PKD激活，以及体内PKD控制的功能表征。秀丽隐杆线虫和哺乳动物共享保守的信号机制、分子和途径，因此，对秀丽隐杆线虫pkd的表征可以深入了解适用于所有真核生物pkd的调控和功能。秀丽隐杆线虫DKF-1 （D激酶家族-1）包含tandemDAGbinding （C1）模块、PH （pleckstrin同源）结构域和Ser/Thr蛋白激酶片段，它们与经典pkd结构域同源。DKF-1和pkd具有相似的底物特异性。Phorbol 12-肉豆蔻酸酯13-乙酸酯（PMA）通过促进激活环中单个氨基酸Thr（588）的磷酸化，原位开关DKF-1的催化活性。当PKC活性被抑制剂消除时，DKF-1的磷酸化和激活不受影响。通过用Ala代替Thr（588）或Gln代替Lys(455)（“激酶死亡”）或用蛋白磷酸酶2C孵育，DKF-1的磷酸化和激酶活性都被消除。因此，DKF-1是一种pma激活的、不依赖于pkc的D激酶。在体内，dkf-1基因启动子活性在神经元中是明显的。dkf-1基因破坏（零表型）和RNA干扰介导的dkf-1蛋白耗竭都会导致下半身瘫痪。靶向表达DKF-1纠正了DKF-1缺失动物的这种运动缺陷。DKF-1的超生理表达将秀丽隐杆线虫的生长限制在正常长度的60%左右。
Protein kinase D (PKD) isoforms are protein kinase C (PKC) effectors in diacylglycerol (DAG)-regulated signaling pathways. Key physiological processes are placed under DAG control by the distinctive substrate specificity and intracellular distribution of PKDs. Comprehension of the roles of PKDs in homeostasis and signal transduction requires further knowledge of regulatory interplay among PKD and PKC isoforms, analysis of PKC-independent PKD activation, and characterization of functions controlled by PKDs in vivo. Caenorhabditis elegans and mammals share conserved signaling mechanisms, molecules, and pathways Thus, characterization of the C. elegans PKDs could yield insights into regulation and functions that apply to all eukaryotic PKDs. C. elegans DKF-1 (D kinase family-1) contains tandemDAGbinding (C1) modules, a PH (pleckstrin homology) domain, and a Ser/Thr protein kinase segment, which are homologous with domains in classical PKDs. DKF-1 and PKDs have similar substrate specificities. Phorbol 12- myristate 13-acetate (PMA) switches on DKF-1 catalytic activity in situ by promoting phosphorylation of a single amino acid Thr(588) in the activation loop. DKF-1 phosphorylation and activation are unaffected when PKC activity is eliminated by inhibitors. Both phosphorylation and kinase activity of DKF-1 are extinguished by substituting Ala for Thr(588) or Gln for Lys(455) ("kinase dead") or incubating with protein phosphatase 2C. Thus, DKF-1 is a PMA-activated, PKC-independent D kinase. In vivo, dkf-1 gene promoter activity is evident in neurons. Both dkf-1 gene disruption (null phenotype) and RNA interference-mediated depletion of DKF-1 protein cause lower body paralysis. Targeted DKF-1 expression corrected this locomotory defect in dkf-1 null animals. Supraphysiological expression of DKF-1 limited C. elegans growth to similar to 60% of normal length.