Tyrosine phosphorylation of the Rab24 GTPase in cultured mammalian cells
Tyrosine phosphorylation of the Rab24 GTPase in cultured mammalian cells
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DOI：
10.1016/j.bbrc.2003.10.171
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发表时间：
2003-12-19
影响因子：
3.1
通讯作者：
Maltese, WA
中科院分区：
文献类型：
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作者：
Ding, J;Soule, G;Maltese, WA
Rab GTP酶大家族的几个成员已被证明在哺乳动物细胞中的囊泡运输中起作用。然而，Rab 24的确切作用仍然不清楚。Rab 24与其他Rab蛋白的不同之处在于，它具有低的内在GT3活性，并且没有被有效地异戊烯化。在这里，我们报告Rab 24的另一个独特性质;即，当在培养的细胞中过表达时，该蛋白质可以经历酪氨酸磷酸化。用特异性抗磷酸酪氨酸单克隆抗体进行免疫印迹分析，结果显示在全细胞裂解物和免疫沉淀样品中mye-Rab 24上存在磷酸酪氨酸（pTyr）。在其他过表达的myc标记的GTP酶（H-Ras、Rab 1b、Rab 6、Rab 11或Rab 13）上未检测到pTyr。来自HEK 293和HEp-2细胞的可溶性和颗粒级分中的myc-Rab 24的比较表明Rab 24的胞质池比膜池更严重地磷酸化。用广谱酪氨酸激酶抑制剂染料木黄酮处理转染细胞。以及特异性Src家族激酶抑制剂PP 2消除了Rab 24的pTyr信号。相反，受体酪氨酸激酶抑制剂，tyrphostin A25，没有效果。Rab 24的酪氨酸磷酸化通过丙氨酸取代两个独特的酪氨酸而减少，一个在高变结构域（Y172）中的强共有磷酸化基序（YXXphi）中发现，另一个落入被称为P环（Y17）的GXXXGK（S/T）基序内。已知后一区域影响Rab蛋白中的GTP水解，因此Y17的磷酸化可能导致Rab 24的低内在GTP酶活性。这是Ras超家族任何成员中酪氨酸磷酸化的首次报道，并且它提出了这种类型的修饰可能影响Rab 24靶向和与效应蛋白复合物的相互作用的可能性。(C)2003年爱思唯尔公司All rights reserved.
Several members of the large family of Rab GTPases have been shown to function in vesicular trafficking in mammalian cells. However, the exact role of Rab24 remains poorly defined. Rab24 differs from other Rab proteins in that it has a low intrinsic GTPase activity and is not efficiently prenylated. Here we report an additional unique property of Rab24; i.e., the protein can undergo tyrosine phosphorylation when overexpressed in cultured cells. Immunoblot analyses with specific anti-phosphotyrosine monoclonal antibodies revealed the presence of phosphotyrosine (pTyr) on mye-Rab24 in whole cell lysates and immunoprecipitated samples. No pTyr was detected on other overexpressed myc-tagged GTPases (H-Ras, Rab1b, Rab6, Rab11 or Rab13). Comparisons of myc-Rab24 in the soluble and particulate fractions from HEK293 and HEp-2 cells indicated that the cytosolic pool of Rab24 was more heavily phosphorylated than the membrane pool. Treatment of transfected cells with the broad-spectrum tyrosine kinase inhibitor, genistein. as well as the specific Src-family kinase inhibitor, PP2, eliminated the pTyr signal from Rab24. In contrast the receptor tyrosine kinase inhibitor, tyrphostin A25, had no effect. Tyrosine phosphorylation of Rab24 was reduced by alanine substitution of two unique tyrosines, one found in a strong consensus phosphorylation motif (YXXphi) in the hypervariable domain (Y172) and the other falling within the GXXXGK(S/T) motif known as the P-loop (Y17). The latter region is known to influence GTP hydrolysis in Rab proteins, so the phosphorylation of Y17 could contribute to the low intrinsic GTPase activity of Rab24. This is the first report of tyrosine phosphorylation in any member of the Ras superfamily and it raises the possibility that this type of modification could influence Rab24 targeting and interactions with effector protein complexes. (C) 2003 Elsevier Inc. All rights reserved.