Cloning, expression, and localization of 230-kDa phosphatidylinositol 4-kinase
Cloning, expression, and localization of 230-kDa phosphatidylinositol 4-kinase
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DOI：
10.1074/jbc.271.20.12088
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发表时间：
1996-05-17
影响因子：
4.8
通讯作者：
Kondo, H
中科院分区：
文献类型：
--
作者：
Nakagawa, T;Goto, K;Kondo, H
从大鼠脑 cDNA 文库中克隆了磷脂酰肌醇 (PI) 4-激酶 cDNA。该cDNA编码2041个氨基酸的蛋白质，计算分子量为231,317。推导的氨基酸序列与两种酵母 PI 4 激酶 STT4 和 PIK1 在假定的催化结构域中分别具有 52.3% 和 34.4% 的同一性，并且在同一结构域中与大鼠 PI 3 激酶的 p110 α 亚基具有 31.7% 的同一性。此外，本发明的cDNA的3'半编码区与最近报道的II型人类PI 4激酶PI4K α的序列有89.6％的同一性，并且其推导的氨基酸序列有98.2％的同一性，这表明PI4K α是本发明的PI 4激酶分子的替代形式。本发明的cDNA含有编码锚蛋白重复结构域、脂质激酶独特结构域、pleckstrin同源结构域、假定的脂质激酶/蛋白激酶同源结构域、富含脯氨酸的区域和SH3结构域的序列。通过表位标签免疫沉淀和常规方法检测转染的COS-7细胞中PI激酶活性，产物磷脂酰肌醇磷酸被鉴定为磷脂酰肌醇4-磷酸，而不是磷脂酰肌醇3-磷酸。 Triton X-100 存在时，PI 4 激酶活性显着增强，但对腺苷的抑制相对不敏感。通过表位标签免疫组织化学，该 PI 4 激酶分子的免疫反应性很大程度上位于与高尔基体囊泡和液泡膜密切相关的位置。通过原位杂交分析，该PI 4激酶的mRNA在整个大脑灰质中明显表达，并且在胎儿大脑中表达强度更高。这些数据表明，这种新型 PI 4 激酶参与了神经元分化和成熟所必需的一些过程，从而避免了突触发生和突触可塑性。
A phosphatidylinositol (PI) 4-kinase cDNA was cloned from a rat brain cDNA library. This cDNA encoded a protein of 2041 amino acids with a calculated molecular weight of 231,317. The deduced amino acid sequence shared the identity of 52.3 and 34.4% in the presumed catalytic domain with two yeast PI 4-kinases, STT4 and PIK1, respectively, and showed 31.7% identity to p110 alpha subunit of rat PI 3-kinase in the same domain. In addition, a 3' half coding region of the present cDNA was 89.6% identical to and its deduced amino acid sequence was 98.2% identical to the sequence for PI4K alpha, a recently reported human PI 4-kinase of type II, suggesting that PI4K alpha is an alternative form of the present PI 4-kinase molecule. The present cDNA contained sequences encoding the ankyrin repeat domain, lipid kinase unique domain, pleckstrin homology domain, presumed lipid kinase/protein kinase homology domain, proline-rich region, and SH3 domain. By examining PI kinase activity in transfected COS-7 cells using the epitope tag immunoprecipitation as well as the conventional way, the product phosphatidylinositol phosphate was identified as phosphatidylinositol 4-phosphate but not phosphatidylinositol 3-phosphate. This PI 4-kinase activity was markedly enhanced in the presence of Triton X-100 but relatively insensitive to inhibition by adenosine. By epitope tag immunohistochemistry, the immunoreactivity for this PI 4-kinase molecule was largely localized in close association with the membranes of the Golgi vesicles and vacuoles. By in situ hybridization analysis, the expression of mRNA for this PI 4-kinase was evident throughout the gray matter of entire brain with higher expression intensity in fetal brain. These data imply that this novel PI 4-kinase is involved in some processes essential to neuronal differentiation and maturation in eluding the synaptogenesis and synaptic plasticity.