Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D
Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D
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DOI：
10.1074/jbc.m005261200
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发表时间：
2000-12-22
影响因子：
4.8
通讯作者：
Schiavo, G
中科院分区：
文献类型：
--
作者：
Iglesias, T;Cabrera-Poch, N;Schiavo, G
蛋白激酶D（PKD）是一种受甘油二酯信号通路调控的丝氨酸/苏氨酸激酶，具有独特的结构域组成和酶学性质，其特异性底物尚待鉴定。在这里，我们已经分离，克隆，并从PC 12细胞，称为Kidins 220（激酶D相互作用底物的220 kDa），作为第一个确定的PKD生理底物的新蛋白质的特征。Kidins 220在N端区域内含有11个锚蛋白重复序列和4个跨膜结构域。我们已经表明，Kidins 220是一种选择性表达于脑和神经内分泌细胞的完整膜蛋白，它集中在神经突的尖端。在PC 12细胞中，PKD共免疫沉淀和磷酸化内源性Kidins 220。在体内通过佛波醇-12，13-二丁酸酯处理刺激PKD活性后，这种磷酸化增加。组成型活性PKD突变体（PKD-S744 E/S748 E）在体外在不存在佛波醇-12，13-二丁酸酯的情况下磷酸化重组Kidins 220-VSVG。相反，当使用PHD的显性负突变体（PKD-D 733 A）时，Kidins 220-VSVG磷酸化被消除。此外，Kidins 220序列内的肽，在PHD特异性磷酸化的共有基序中含有丝氨酸919，表现为迄今为止最好的肽底物。丝氨酸919取代丙氨酸废除肽磷酸化。此外，通过产生识别丝氨酸919上磷酸化的Kidins 220的抗体，我们表明佛波酯处理导致体内PC 12细胞中该残基的特异性磷酸化。我们的结果提供了PKD的第一个生理底物，并表明Kidins 220在体内被PKD在丝氨酸919处磷酸化。
Protein kinase D (PKD) is a serine/threonine kinase regulated by diacylglycerol signaling pathways with unique domain composition and enzymatic properties, still awaiting identification of its specific substrate(s). Here we have isolated, cloned, and characterized a novel protein from PC12 cells, termed Kidins220 (kinase D-interacting substrate of 220 kDa), as the first identified PKD physiological substrate. Kidins220 contains 11 ankyrin repeats and four transmembrane domains within the N-terminal region. We have shown that Kidins220 is an integral membrane protein selectively expressed in brain and neuroendocrine cells, where it concentrates at the tip of neurites. In PC12 cells, PKD coimmunoprecipitates and phosphorylates endogenous Kidins220. This phosphorylation is increased after stimulating PKD activity in vivo by phorbol-12,13-dibutyrate treatment. A constitutively active PKD mutant (PKD-S744E/S748E) phosphorylates recombinant Kidins220-VSVG in vitro in the absence of phorbol-12,13-dibutyrate. Conversely, Kidins220-VSVG phosphorylation is abolished when a dominant negative mutant of PHD (PKD-D733A) is used. Moreover, a peptide within the Kidins220 sequence, containing serine 919 in a consensus motif for PHD-specific phosphorylation, behaved as the best peptide substrate to date. Substitution of serine 919 to alanine abrogated peptide phosphorylation. Furthermore, by generating an antibody recognizing Kidins220 phosphorylated on serine 919, we show that phorbol ester treatment causes the specific phosphorylation of this residue in PC12 cells in vivo. Our results provide the first physiological substrate for PKD and indicate that Kidins220 is phosphorylated by PKD at serine 919 in vivo.