The regulation mechanism of lipid kinase in the signal transduction

脂质激酶在信号转导中的调控机制

• 批准号: 09044248
• 负责人: KONDO Hisatake
• 金额: $ 10.11万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044248/
• 关键词: lipid kinase; PI-signaling; FABP; CDP-DG synthase; gene knockout; in situ hybridization; brain; PI3-キナーゼ; PI4-キナーゼ; PLD; 脳内発現局在; ジアシルグリセロールキナーゼ; PI3キナーゼ; 遺伝子クローニング; ゲノム解析; 脂肪酸結合蛋白

A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this CDS was 53 kDa, and putative primary structure included several possible membrane-spanning regions. This enzyme molecule preferred 1-stearoyl- 2-arachidonoyl phosphatidate as a substrate and its activity was strongly inhibited by phosphatidylinositol (PtdIns) 4,5-bisphosphate. The molecule was mainly localized in close association with the membrane of the endoplasmic reticulum of over-expressed in vitro cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, pineal body, and the inner segment of photoreceptor cells, and postmitotic spermatocytes and spermatids of testis of mature rats. This finding is closely similar to that of arachidonoyl-diacylglycerol kinase (DGK) and in this regard we discussed with Dr.J.A.Glomset who first id … More entified the DGK and attempted to examine the colocalization of the two molecules by immunohistochemistry by Drs.K.Goto.A novel DGK was identified by cDNA cloning from human brain and retina cDNA library and termed DGKtheta in co-operation with Dr.W.J.van Blitterswijk. DGKtheta was composed of 951 amino acids and contained three cysteine-rich domain, a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. In adult rat brain high expression was detected in the cerebellar cortex and hippocampus.The cloning and characterization of a novel class II phosphoinositide 3-kinase was succeeded from the cDNA library of regenerating rat liver. This enzyme molecule composed of1505 amino acids contained a C2 domain and displayed a restricted substrate specificity for PtdIns and PtdIns 4-P and, thus termed PI3K-IIgamma. This molecule was localized in the juxtanuclear Golgi region in the over-expressed in vitro cells. The mRNA expression was confined to the liver throughout the development and its expression increased during liver regeneration after partial hepatectomy. Further molecular characterization is under way in co-operation with Dr.L.C.Cantley.By in situ hybridization histochemistry a dramatic increase in expression of Akt was detected in affected hypoglossal nucleus after 2-7 days following axotomy of the hypoglossal nerve. Such an increase in the gene expression after axotomy was observed in a similar time course for PI3 kinase and skin type-fatty acid binding protein (FABP), suggesting a functional relation between PI-signal and FABP.Thus the gene knockout for s-FABP is under way in co-operation with Dr.F.Spener.The gene expression localization of phospholipase D in the brain is also under way in cooperation with Dr.Y.Nozawa. The chromosome mapping of the genome for PtdIns 4-kinase was attempted in cooperation with Dr.D.M.Hunt and started. However, in the course of experiment we unfortunately found that it has already done by another foreign research group and thus gave up this attempt. Less

从大鼠脑cDNA文库中首次克隆到一个编码462个氨基酸的蛋白质，该蛋白质具有CDP-二酰甘油合成酶（CDS）活性，是脊椎动物CDS酶分子。该CDS的推导分子量为53 kDa，推定的一级结构包括几个可能的跨膜区域。该酶分子优选1-硬脂酰-2-花生四烯酸磷脂酸作为底物，其活性被磷脂酰肌醇（PtdIns）4，5-二磷酸强烈抑制。该分子主要定位于与过表达的体外细胞的内质网膜紧密结合。CDS mRNA的强表达定位于小脑浦肯野细胞、松果体、光感受器细胞内段和成熟大鼠睾丸有丝分裂后的精母细胞和精子细胞。这一发现与花生四烯酰甘油二酯激酶（DGK）的发现非常相似，在这方面，我们与J.A.Glomset博士进行了讨论，他首先提出了 ...更多信息 K. Goto博士鉴定了DGK，并试图通过免疫组织化学方法检测这两种分子的共定位，与W. J.货车Blitterswijk博士合作，通过从人脑和视网膜cDNA文库中克隆cDNA鉴定了一种新的DGK，并命名为DGKtheta。DGKtheta由951个氨基酸组成，包含三个富含半胱氨酸的结构域、一个富含脯氨酸和甘氨酸的结构域和一个推测的SH 3结构域结合位点以及一个具有重叠Ras结合结构域的普列克底物同源结构域。在成年大鼠脑组织中，在小脑皮质和海马中有高表达，并从再生大鼠肝cDNA文库中成功地克隆了一个新的II类磷酸肌醇3-激酶。该酶分子由1505个氨基酸组成，含有C2结构域，对PtdIns和PtdIns 4-P具有限制性底物特异性，因此被称为PI 3 K-II γ。该分子在体外过表达的细胞中定位于非核高尔基体区域。mRNA的表达仅限于肝脏的整个发展和表达的肝部分切除后的肝再生过程中增加。与Dr.L.C.Cantley合作正在进行进一步的分子表征。通过原位杂交组织化学，在舌下神经轴突切断术后2-7天，在受影响的舌下神经核中检测到Akt表达的显著增加。在神经切断后，PI 3激酶和皮肤型脂肪酸结合蛋白（FABP）的基因表达在类似的时间过程中增加，这表明PI信号和FABP之间存在功能性关系。因此，S-FABP的基因敲除正在与F. Szawa博士合作进行。磷脂酶D在脑中的基因表达定位也正在与Y. Nozawa博士合作进行。与Dr.D.M.Hunt合作尝试并开始PtdIns 4-激酶基因组的染色体作图。然而，在实验过程中，我们不幸地发现，它已经由另一个外国的研究小组，从而放弃了这一尝试。少

项目成果

H.Kondo et al.: "A novel class II phosphoinositide 3 kinase predominantly expressed in the liver" J.Biol.Chem.273・13. 7731-7736 (1998)

H.Kondo 等人：“一种主要在肝脏中表达的新型 II 类磷酸肌醇 3 激酶”J.Biol.Chem.273·13 (1998)。

H.Kondo: "Localization of wRNA for fatty aeid fransporter protein in developag and nature brain of rats" Mol. Brain Res.46(1). 217-222 (1997)

H.Kondo：“大鼠发育和自然大脑中脂肪酸转运蛋白的 wRNA 定位”Mol。

H.Kondo et al.: "A novel class II phosphoinositide 3-kinase predominantly expressed in the liver and enhanced expression Luring livew wegeneration" J.Biol.Chem.273,13. 7731-7736 (1998)

H.Kondo 等人：“一种新型 II 类磷酸肌醇 3-激酶，主要在肝脏中表达，并增强表达引诱活体一代”J.Biol.Chem.273,13。

H.Kondo: "Expression of wRNA for AKT, serine-threonine protein kinase in the brain guny development and its transient enhancemant following axotomy of hypoglossal nave" J. Mol. Neurosci. 9(1). 27-33 (1997)

H.Kondo：“AKT、丝氨酸-苏氨酸蛋白激酶的 wRNA 在脑群发育中的表达及其在舌下中殿轴切术后的短暂增强”J. Mol。

Suzuki, R., Sakagami, H.& Kondo, H.: "Localization of mRNA for Dri 42, a subtype 2b of phosphatidic acid phosphatase, in developing and mature brain of rats." Mol.Brain.Res.(in press).

铃木，R.，坂上，H.