Molecular complexes that contain both c-Cbl and c-Src associate with Golgi membranes
Molecular complexes that contain both c-Cbl and c-Src associate with Golgi membranes
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DOI：
10.1078/0171-9335-00217
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发表时间：
2002-01-01
影响因子：
6.6
通讯作者：
Baron, R
中科院分区：
文献类型：
--
作者：
Bard, F;Patel, U;Baron, R
Cbl 是一种衔接蛋白，在多种受体和非受体酪氨酸激酶激活后被磷酸化并募集到这些激酶上。与激活的受体结合后，Cbl 作为激酶抑制剂和 E3 泛素连接酶发挥关键作用，从而有助于受体下调和内化。此外，受体激活后，Cbl 易位至细胞内囊泡区室。我们在此报道 Cbl 还与高尔基膜相关。对多种未刺激细胞（包括 CHO 细胞）中的 Cbl 进行共聚焦免疫荧光染色，揭示了 Cbl 和高尔基体标记的显着核周共定位。显着的 Cbl 染色和高尔基体标记均被布雷菲德菌素 A 分散。CHO 细胞的亚细胞分级分离表明，约 10% 的 Cbl 与膜稳定结合，并且相对于其他膜组分，通过等密度离心和自由流动电泳产生的富含高尔基体的膜组分也富含 Cbl。膜结合的 Cbl 被过度磷酸化，并与内源性 Src 进行免疫共沉淀。通过免疫荧光，一些 Src 与 Cbl 和高尔基体标记共定位，并且 Src 与 Cbl 一样，存在于通过顺序密度离心和自由流动电泳制备的高尔基体富集级分中。转染活化形式的 Src（而非野生型 Src）会增加与 Cbl 免疫共沉淀的 Src 量，并增加高尔基体上 Cbl 染色的强度。这一结果与膜相关 Cbl 酪氨酸磷酸化的增加一起表明，高尔基体相关 Cbl 可能是包含活化 Src 的分子复合物的一部分。 Src和Cbl在高尔基体的定位和相互作用以及Cbl与高尔基体膜相互作用的调节表明该复合物可能有助于高尔基体功能的调节。
Cbl is an adaptor protein that is phosphorylated and recruited to several receptor and non-receptor tyrosine kinases upon their activation. After binding to the activated receptor, Cbl plays a key role as a kinase inhibitor and as an E3 ubiquitin ligase, thereby contributing to receptor down-regulation and internalization. In addition, Cbl translocates to intracellular vesicular compartments following receptor activation. We report here that Cbl also associates with Golgi membranes. Confocal immunofluorescence staining of Cbl in a variety of unstimulated cells, including CHO cells, revealed a prominent perinuclear colocalization of Cbl and a Golgi marker. Both the prominent Cbl staining and the Golgi marker were dispersed by brefeldin A. Subcellular fractionation of CHO cells demonstrated that about 10% of Cbl is stable associated with membranes, and that Golgi-enriched membrane fractions produced by isopycnic density centrifugation and free-flow electrophoresis are also enriched in Cbl, relative to other membrane fractions. The membrane-bound Cbl was hyperphosphorylated and it co-immunoprecipitated with endogenous Src. By immunofluorescence, some Src colocalized with Cbl and Golgi markers, and Src, like Cbl, was present in the Golgi-enriched fraction prepared by sequential density centrifugation and free-flow electrophoresis. Transfection of an activated form of Src, but not wild-type Src, increased the amount of Src that co-immunoprecipitated with Cbl, and increased the intensity of Cbl staining on the Golgi. This result, together with the increased tyrosine phosphorylation of the membrane-associated Cbl, suggests that Golgi-associated Cbl could be part of a molecular complex that contains activated Src. The localization and interaction of Src and Cbl at the Golgi and the regulation of the interaction of Cbl with Golgi membrane suggest that this complex may contribute to the regulation of Golgi function.