Characterization and location of Src-dependent tyrosine phosphorylation in rat brain mitochondria
Characterization and location of Src-dependent tyrosine phosphorylation in rat brain mitochondria
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DOI：
10.1016/s0167-4889(02)00174-x
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发表时间：
2002-04-03
影响因子：
5.1
通讯作者：
Toninello, A
中科院分区：
文献类型：
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作者：
Salvi, M;Brunati, AM;Toninello, A
在高度纯化的大鼠脑线粒体中的蛋白质磷酸化的分析显示，存在几种碱稳定的磷蛋白，其磷酸化显着增加后，治疗与过氧钒酸盐和Mn 2+，一个属性，表明酪氨酸磷酸化。这些包括三个明显的条带，其表观大小为50、60和75 kDa，可通过抗磷酸酪氨酸检测。酪氨酸磷酸化消失时，线粒体与PP 2，Src激酶家族的抑制剂处理，这表明该家族的成员在大鼠脑线粒体的存在。线粒体裂解物的免疫印迹和免疫沉淀测定证实了Fyn、Src和林恩激酶以及Csk（一种负控制Src激酶家族活性的蛋白激酶）的存在。结果表明，酪氨酸磷酸化的蛋白质是膜结合的，并且它们位于外膜的内表面和/或内膜的外表面上。相反，Src酪氨酸激酶主要位于膜间隙-特别是，如林恩激酶的免疫金实验所揭示的，在嵴腔中。大鼠脑线粒体也被发现具有显着水平的酪氨酸磷酸酶活性，强烈抑制过钒酸盐。(C)2002 Elsevier Science B. V.保留所有权利。
Analysis of protein phosphorylation in highly purified rat brain mitochondria revealed the presence of several alkali-stable phosphoproteins whose phosphorylation markedly increases upon treatment with peroxovanadate and Mn2+, a property indicating tyrosine phosphorylation. These include three prominent bands, with apparent sizes of 50, 60, and 75 kDa, which are detectable by anti-phosphotyrosine. Tyrosine phosphorylation disappears when mitochondria are treated with PP2, an inhibitor of the Src kinase family, suggesting the presence of members of this family in rat brain mitochondria. Immunoblotting and immunoprecipitation assays of mitochondrial lysates confirmed the presence of Fyn, Src and Lyn kinases, as well as Csk, a protein kinase which negatively controls the activity of the Src kinase family. Results show that tyrosine-phosphorylated proteins are membrane-bound and that they are located on the inner surface of the outer membrane and/or the external surface of the inner membrane. Instead, Src tyrosine kinases are mainly located in the intermembrane space - in particular, as revealed by immunogold experiments for Lyn kinase, in the cristal lumen. Rat brain mitochondria were also found to possess a marked level of tyrosine phosphatase activity, strongly inhibited by peroxovanadate. (C) 2002 Elsevier Science B.V. All rights reserved.