| Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
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Author:
Date:
2016-10-05
[Abstract] This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of ...
[摘要] 该协议描述了通过激酶PINK1使用重组蛋白的泛素和帕金蛋白的体外磷酸化。两种底物，泛素和帕金蛋白，在保守的丝氨酸65残基（pS65-泛素和pS65-帕金蛋白）上磷酸化。该方案还包括使用单体和K48和K63连接的聚泛素链作为替代底物。虽然有市售的抗体，我们没有测试他们在这个测定中的性能，因为，但使用我们实验室的验证抗体。另外描述了另一种抗体非依赖性方法，使用phos-标记凝胶检测pS65-泛素和pS65-帕金。
[背景] 在细胞中， PINK1是稳定和激活的线粒体膜去极化和其他形式的应力，导致线粒体损伤。活化的PINK1磷酸化泛素，其作为线粒体表面上胞质E3泛素连接酶Parkin的受体。 Parkin对PINK1的磷酸化是Parkin对线粒体底物的完全活性所必需的。活性pS65-Parkin的存在在前馈机制中扩增了作为线粒体标记的线粒体上的pS65-泛素的量。最终，受损的线粒体被自噬噬菌体衔接子识别，并将被蛋白酶体和自噬（mitophagy）降解。这种关键的线粒体质量控制通路促进线粒体的周转，并防止可导致细胞变性的功能障碍线粒体的积累。 PINK1或Parkin中的功能缺失突变与早发性帕金森病相关。
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| Detection of Phospho-KRAS by Electrophoretic Mobility Change in Human Cell Lines and in Tumor Samples from Nude Mice Grafts
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Author:
Date:
2015-03-20
[Abstract] KRAS is the oncogene most frequently mutated in human solid tumors especially in pancreas, colon, small intestine, biliary tract and lung. We have recently demonstrated that oncogenic KRAS needs S181 phosphorylation to fully display its oncogenic features suggesting its inhibition as a therapeutic treatment against KRAS-driven tumors. Due to the importance to detect KRAS phosphorylation in human tumors and the absence of specific antibodies against phosphorylated KRAS, we developed a new protocol based on the Phos-tag SDS methodology to detect this post-translational modification for KRAS. ...
[摘要] KRAS是在人实体瘤特别是在胰腺，结肠，小肠，胆道和肺中最常突变的癌基因。 我们最近证明，致癌KRAS需要S181磷酸化充分显示其致癌特征，表明其作为对KRAS驱动的肿瘤的治疗性治疗的抑制作用。 由于在人类肿瘤中检测KRAS磷酸化的重要性和不存在针对磷酸化KRAS的特异性抗体，我们开发了基于Phos-tag SDS方法的新方案以检测这种KRAS的翻译后修饰。 Phos标签是特异性结合磷酸化蛋白质的分子，降低其在SDS-PAGE中的迁移速度并允许其从非磷酸化形式分离。
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| Phosphatase Protection Assay: 14-3-3 Binding Protects the Phosphate group of RSG from λ Protein Phosphatase
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Author:
Date:
2015-02-05
[Abstract] 14-3-3 proteins regulate diverse cellular processes in eukaryotes by binding to phospho-serine or threonine of target proteins. One of the physiological functions of 14-3-3 is to bind and protect phosphate groups of the target proteins against phosphatases. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in the feedback regulation of biosynthetic genes of plant hormone gibberellin. 14-3-3 binds to phospho-Ser-114 in RSG. Ca2+-dependent protein kinase NtCDPK1 was identified as a kinase that phosphorylates Ser-114 of ...
[摘要] 14-3-3蛋白通过结合靶蛋白的磷酸 - 丝氨酸或苏氨酸来调节真核生物中的多种细胞过程。 14-3-3的生理功能之一是结合和保护靶蛋白的磷酸基团免受磷酸酶。生长生长的表达（RSG）是涉及植物激素赤霉素的生物合成基因的反馈调节的烟草（烟草属）转录因子。 14-3-3结合RSG中的磷酸-Ser-114。 Ca 2+ - 依赖性蛋白激酶NtCDPK1被鉴定为磷酸化RSG的Ser-114的激酶。我们最近的研究揭示NtCDPK1与RSG和14-3-3形成异源三聚体，并且14-3-3从NtCDPK1转移到磷酸化RSG（Ito等人，2014）。在研究过程中，我们发现14-3-3在体外保护RSG的磷酸基团免受λ蛋白磷酸酶的影响。在这里，我们描述了体外磷酸酶保护测定的协议。为了检测蛋白质的磷酸化状态，我们使用Phos-tag SDS-PAGE和放射自显影。该方案可以适用于磷酸蛋白结合蛋白是否保护靶蛋白的磷酸基团免于磷酸酶的检查，尽管蛋白激酶可能是靶蛋白磷酸化所需的。
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