4种哺乳动物抑制素亚型中只有1种,arrestin-3促进c-JunN末端激酶(JNK)家族激酶的激活。这里,我们描述了两组不同的协议,用于阐明所涉及的机制。一种是基于来自以下纯化蛋白的信号传导模块的重建:抑制蛋白-3、MKK4、MKK7、JNK1、JNK2和JNK3。该方法的主要优点是它明确地确定哪些作用是直接的,因为在这些测定中仅存在预期的纯化蛋白质。主要缺点是这些级联的最上游激酶,ASK1或其他MAP3K,没有纯化的形式,限制重建不完整的双激酶模块。另一种方法用于分析抑制蛋白-3对完整细胞中JNK活化的影响。在这种情况下,信令模块包括ASK1和/或其他MAP3K。然而,因为每个细胞都表达数千种不同的蛋白质,不能排除它们对读数的可能影响。尽管如此,从纯化的蛋白质体外重建和基于细胞的测定的组合使得阐明JNK家族激酶的抑制蛋白-3依赖性激活的机制成为可能。©2023威利期刊有限责任公司。基本方案1:使用纯化的蛋白质在体外构建抑制蛋白-3支架MKK4/7-JNK1/2/3信号传导模块替代方案1:通过使用抗磷酸-JNK抗体支持免疫印迹测量JNK1/2磷酸化,MKK4/7对抑制蛋白-3介导的JNK1/2激活的表征方案1:表达,净化,和激活GST-MKK4支持协议2:表达式,净化,和激活GST-MKK7-His6支持协议3:表达,净化,和无标签JNK1Α1支持协议4的激活:表达,净化,和无标签JNK2Α2的活化基本方案2:分析抑制蛋白-3在完整细胞中ASK1/MKK4/MKK7诱导的JNK活化中的作用替代方案2:分析抑制蛋白-3在完整细胞中MKK4诱导的JNK活化中的作用基本方案3:表征抑制蛋白-3对ASK1/MK7刺激的JNK细胞磷酸化的双相作用。
Only 1 out of 4 mammalian arrestin subtypes, arrestin-3, facilitates the activation of c-Jun N-terminal kinase (JNK) family kinases. Here, we describe two different sets of protocols used for elucidating the mechanisms involved. One is based on reconstitution of signaling modules from the following purified proteins: arrestin-3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it unambiguously establishes which effects are direct because only intended purified proteins are present in these assays. The key drawback is that the upstream-most kinases of these cascades, ASK1 or other MAP3Ks, are not available in purified form, limiting reconstitution to incomplete two-kinase modules. The other approach is used for analyzing the effects of arrestin-3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAP3Ks. However, as every cell expresses thousands of different proteins, their possible effects on the readout cannot be excluded. Nonetheless, the combination of in vitro reconstitution from purified proteins and cell-based assays makes it possible to elucidate the mechanisms of arrestin-3-dependent activation of JNK family kinases. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Construction of arrestin-3-scaffolded MKK4/7-JNK1/2/3 signaling modules in vitro using purified proteins Alternate Protocol 1: Characterization of arrestin-3-mediated JNK1/2 activation by MKK4/7 by measurement of JNK1/2 phosphorylation using immunoblotting with anti-phospho-JNK antibody Support Protocol 1: Expression, purification, and activation of GST-MKK4 Support Protocol 2: Expression, purification, and activation of GST-MKK7-His6 Support Protocol 3: Expression, purification, and activation of tagless JNK1Α1 Support Protocol 4: Expression, purification, and activation of tagless JNK2Α2 Basic Protocol 2: Analysis of the role of arrestin-3 in ASK1/MKK4/MKK7-induced JNK activation in intact cells Alternate Protocol 2: Analysis of the role of arrestin-3 in MKK4-induced JNK activation in intact cells Basic Protocol 3: Characterization of the biphasic effect of arrestin-3 on ASK1/MKK7-stimulated JNK phosphorylation in cells.