CD44-ERM-ACTIN SYSTEM,SIGNAL TRANSDUCTION,AND APOPTOSIS

CD44-ERM-肌动蛋白系统、信号转导和细胞凋亡

• 批准号: 07458189
• 负责人: TSUKITA Sachiko
• 金额: $ 4.74万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458189/
• 关键词: CD44; EZRIN; RADIXIN; MOESIN; RHO; APOTOSIS; ACTIN FILAMENT; RHO-GDI; ERM; アクチン

The ERM proteins, ezrin, radixin and moesin, are involved in actin filament/plasma membrane interaction as crosslinkers. CD44 was identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the GST/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, at low ionic strength ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3(]SY.+-。[)1.6nM), but with low affinity at physiological ionic strength. However, in the presence of phosphoinositides (PI,4-PIP,and 4,5-PIP<@D22@>D2), ERM proteins bound with relatively high affinity to GST-CD44cyt even at physiological ionic strength : 4,5-PIP<@D22@>D2 showed the most remarkable effect (Kd of moesin in the presence of 4,5-PIPP<@D22@>D2 was 9.3(]SY … More .+-。[)4.8nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDI,a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTPgammaS,and remarkedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP-form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also remarkably suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, when living BHK cells were treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM.These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover is possibly involved in this regulation mechanism. Less

ERM蛋白Ezrin、Radioxin和Moesin作为交联剂参与肌动蛋白细丝/质膜的相互作用。CD44被认为是ERM蛋白的主要膜结合伙伴之一。为了研究CD44/ERM蛋白在体外的相互作用，我们通过重组杆状病毒感染的方法制备了小鼠Ezrin、Radioxin、moesin和GST/CD44胞浆结构域融合蛋白(GST-CD44cyt)，并建立了ERM蛋白与CD44胞浆结构域结合的体外实验。在该体系中，在低离子强度下，ERM蛋白与GST-CD44cyt高亲和力结合(moesin的Kd值为9.3(]sy.+)。[)1.6 nm)，但在生理离子强度下亲和力较低。然而，在磷脂酰肌醇(PI，4-PIP和4，5-PIP；@D22@&GT；D2)存在下，即使在生理离子强度4，5-PIP@D22@&GT；D2时，与GST-…结合的ERM蛋白的亲和力也相对较高，表现出最显著的作用(4，5-PIPP；@D22@&GT；D2在4，5-PIPP&lt；@D22@&GT；D2存在下的Kd值为9.3更多。+-。[)4.8 nm)。接下来，为了研究CD44/ERM相互作用在体内的调节机制，我们重新检测了BHK细胞免疫沉淀的CD44/ERM复合体，发现它含有Rho GTP酶的调节因子Rho-GDI。然后，我们评估了Rho参与CD44/ERM复合体形成的调节。将重组ERM蛋白与培养的BHK细胞裂解液孵育，离心后，部分重组ERM蛋白被回收到不溶性部分。这种结合被GTPGammaS增强，并被Rho的特异性抑制剂C3毒素显著抑制，表明这种结合需要裂解物中的GTP形式的Rho。针对CD44胞浆区域的单抗也显著抑制了这种结合，将不溶部分中的大多数外源ERM蛋白的结合伙伴鉴定为CD44。与这一结合分析一致的是，当活的BHK细胞被C3毒素处理时，大多数不溶的ERM蛋白移动到细胞质中的可溶室中，使CD44远离ERM。这些发现表明，Rho调节体内CD44/ERM复合体的形成，磷脂酰肌醇的转化可能参与了这一调节机制。较少

项目成果

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