Intracellular distribution and regulatory function of protein phosphatases

蛋白磷酸酶的细胞内分布和调节功能

• 批准号: 09670042
• 负责人: TAKAI Akira
• 金额: $ 0.38万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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-09670042/
• 关键词: Protein phosphatases; Protein phosphorylation; Signal transduction; Okadaic acid; Microcystins; Tautomycin; CRTR; Ciliary muscle; ト-トマイシン; 酵素阻害剤

The aim of this project has been to investigate the roles of reversible protein phosphorylation in regulation of cell motility and ion transport. During the term of this project we carried out physiological and biochemical experiments chiefly in smooth and cardiac muscles, as summarized below. Part of the results obtained have been published (see the reprints attached).(1) Okadaic acid (OA) is a potent inhibitor of protein phosphatase 1 and 2A (PP1 and PP2A) having a higher affinity to PP2A than to PP1. In smooth muscle preparations with intact plasma membrane, OA reversibly inhibits contraction and myosin light-chain (MLC) phosphorylation. Another PP inhibitor, tautomycin, which exhibits a higher affinity PP1 than PP2A is known to enhance contraction and myosin light chain phosphorylation. In the present experiments we have observed that OA strongly inhibits this contractile effect of tautomycin. The MLC phosphorylation was also decreased by addition of OA but about 10 % of MLC were s … More till phosphorylated even when contraction was completely inhibited by OA.These results indicate that there are several steps toward smooth muscle contraction which is suppressed by protein phosphorylation.(2) In smooth muscle cells isolated from bovine ciliary body we have studied effects of carbachol on the membrane potential and current using the whole-cell clamp technique. We have demonstrated the existence of a non-selective cation channel which is activated by muscarinic receptors belonging to the M3 subtype. This channel, which admits Ca^<2+>, may serve at least partly as a Ca^<2+> entry required for sustained contraction.(3) In isolated myocyte of guinea-pig ventricle, we have examined the effect of anthracene-9-carboxylic acid (9AC), a Cl^- channel inhibitor, on the CFTR channel. We have shown that the activating effect of isoprenaline or forskolin on the CFTR is reversibly enhanced and prolonged in the presence of 9AC.We have also shown that 9AC inhibits a fraction of intracellular p-nitrophenyl phosphatase activity, which is insensitive to known phosphatase inhibitors, such as OA, tartaric acid or bromotetramisole. Less

本项目的目的是研究可逆性蛋白磷酸化在调节细胞运动和离子转运中的作用。在本项目期间，我们主要对平滑肌和心肌进行了生理和生化实验，总结如下。(1)冈田酸(OA)是蛋白质磷酸酶1和2 A(PP1和PP2A)的有效抑制剂，与PP2A的亲和力高于对PP1的亲和力。在质膜完整的平滑肌制剂中，OA可逆地抑制收缩和肌球蛋白轻链(MLC)的磷酸化。另一种PP抑制剂，交托霉素，表现出比PP2A更高的亲和力，可以增强收缩和肌球蛋白轻链磷酸化。在目前的实验中，我们观察到OA强烈地抑制了他托霉素的这种收缩作用。OA的加入也降低了巨噬细胞的磷酸化，但约有10%的巨噬细胞是S…这些结果表明，即使在OA完全抑制收缩的情况下，也有可能发生更多的磷酸化。这些结果表明，平滑肌收缩有几个步骤，而这种收缩是被蛋白质磷酸化抑制的。(2)在分离的牛睫状体平滑肌细胞中，我们用全细胞钳技术研究了氨基甲胆碱对膜电位和电流的影响。我们已经证明了非选择性阳离子通道的存在，该通道由属于M3亚型的M受体激活。该通道允许Ca~(2+)进入，至少部分作为持续收缩所需的Ca~(2+)通道。(3)在豚鼠心室肌细胞上，我们观察了氯离子通道阻断剂-9-羧酸(9AC)对CFTR通道的影响。结果表明，在9AC存在下，异丙肾上腺素或Forsklin对CFTR的激活作用可逆地增强和延长。我们还发现，9AC抑制了细胞内的一部分对硝基苯基磷酸酶的活性，这种活性对已知的磷酸酶抑制剂如OA、酒石酸或溴四咪唑不敏感。较少

项目成果

高井 章: "PP1およびPP2Aに特異的阻害作用を有する天然毒素" 蛋白質・核酸・酵素. 43(8). 1091-1101 (1998)

Akira Takai：“对 PP1 和 PP2A 具有特定抑制作用的天然毒素”，蛋白质、核酸和酶 43(8) (1998)。

Yotsu-Yamashita,M., et al.: "Phosphorylated retinoblastoma protein stimulates DNA polymerase α" Journal of Pharmacology and Experimental Therapeutics. 印刷中. (1999)

Yotsu-Yamashita, M. 等人：“磷酸化视网膜母细胞瘤蛋白刺激 DNA 聚合酶 α”，《药理学和实验治疗学杂志》出版（1999 年）。

高井 章: "蛋白質脱燐酸化反応:解析の基本手技" 日本血栓止血学会誌. 8(6). 504-516 (1997)

Akira Takai：“蛋白质去磷酸化反应：分析基本技术”日本血栓和止血学会杂志 8(6) (1997)。

Takemura,M.,Kitagawa,et al.: "Phosphorylated retinoblastoma protein stimulates DNA polymerase α" Oncogene. 15. 2483-249〓 (1997)

Takemura, M., Kitakawa, et al.：“磷酸化视网膜母细胞瘤蛋白刺激 DNA 聚合酶 α”Oncogene 15. 2483-249〓 (1997)

TAKEMURA,M., KITAGAWA,T., IZUTA,S., WASA,J., TAKAI,A., AKIYAMA,T.& YOSHIDA,S.: "Phosphorylated retinoblastoma protein stimulates DNA polymerase alpha." Oncogene. 15. 2483-2492 (1997)

竹村，M.，北川，T.，井田，S.，WASA，J.，高井，A.，秋山，T.