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

cDNA编码了462-氨基酸蛋白，该蛋白显示CDP二酰甘油合酶（CDS）活性首次克隆为大鼠脑cDNA库中的脊椎动物酶分子。该CDS的分子质量为53 kDa，推定的主要结构包括几个可能的膜跨度区域。该酶分子首选1稳态-2-芳基的磷脂酸作为底物，其活性受到磷脂酰肌醇（PTDINS）4,5-双磷酸的强烈抑制。该分子主要与过表达的体外细胞内质网的膜密切相关。 CD的强烈mRNA表达位于小脑Purkinje细胞中，松果体和感光细胞的内部段，以及成熟大鼠睾丸的有丝分裂后精子细胞和精子。这一发现与Arachidonoyl-二酰基甘油激酶（DGK）非常相似，在这方面，我们与J.A.Glomet的博士讨论了，谁首先ID…更确定了DGK，并试图检查了由cdne and reted dgn and reted dgn and dgn cdn and reted dgn cdn and retive cdn and ret dgk vish.a and ret tern cdn and cdne dgn cdn and ret dgk ccdne and reted clone and cdne and reted clone and dgn。 DGKTHETA与W.J.Van Blitterswijk博士合作。 DGKTHETA由951个氨基酸组成，并包含三个富含半胱氨酸的结构域，一个带有推定的SH3结构域结合位点的脯氨酸和富含甘氨酸的结构域和一个带有重叠的RAS RAS社会联合域的Pleckstrin同源域。在成年大鼠脑中，在小脑皮层和海马中检测到高表达。新型II类磷酸肌醇3-激酶的克隆和表征从再生大鼠肝脏的cDNA库中成功。该酶分子由1505个氨基酸组成，其中包含一个C2结构域，并显示了PTDINS和PTDINS 4-P的受限底物特异性，因此称为PI3K-IIGAMMA。该分子位于过表达的体外细胞中的近核高尔基区。在整个发育过程中，mRNA表达局限于肝脏，部分肝后肝脏再生期间的表达增加。在与原位杂交组织化学的合作中，进一步的分子表征正在与L.C. cantley。在类似的PI3激酶和皮肤类型脂肪酸结合蛋白（FABP）的时间过程中观察到轴切开术后基因表达的这种增加，这表明PI-信号和FABP之间的功能关系。因此，S-FABP的基因敲除与基因iSpasase的合作中，S-FABP的基因敲除在磷酸化学的局部化中也在合作中。 Y.Nozawa博士。试图与D.M.亨特博士合作并开始，尝试了PTDINS 4-激酶基因组的染色体映射。但是，在实验过程中，我们不幸发现它已经由另一个外国研究小组完成，因此放弃了这一尝试。较少的

项目成果

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" 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: "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。

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。

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.