Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells
Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells
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DOI：
10.1111/j.1432-1033.1997.t01-1-00660.x
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发表时间：
1997-09-15
期刊：
影响因子：
--
通讯作者：
Maenhaut, C
中科院分区：
文献类型：
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作者：
Wilkin, F;SuarezHuerta, N;Maenhaut, C
我们已经分离出编码一种新蛋白质的狗和人形式的cDNA克隆，该蛋白质的功能仍然未知。序列分析表明，犬克隆c5 fw蛋白含有343个氨基酸残基。几个潜在的磷酸化位点，以及12个保守的亚结构域中的两个（VIII和IX），它们折叠成蛋白激酶大家族的共同催化核心结构。人克隆c5 fw与其dug对应物具有95%的氨基酸同源性，我们还分离了另一个人相关克隆c5 fw，其与狗序列具有70%的氨基酸同源性。我们在COS-7细胞中瞬时表达c-myc表位标记的克隆c5 fw蛋白，并用含有克隆c5 fw cDNA的重组腺病毒（腺病毒c5 fw）感染原代培养的甲状腺细胞。在这两个实验中，检测到46 kDa的蛋白质，随后更广泛的特点。通过双向电泳和V8蛋白酶消化，我们发现，这种过表达的蛋白质是磷酸化的不同位点。此外，用促甲状腺素或表皮生长因子促甲状腺素和胎牛血清刺激的细胞增加了含有克隆c5 fw的腺病毒感染后产生的克隆c5 fw蛋白的水平。嘌呤霉素处理1小时后该46-kDa蛋白的消失表明其为不稳定蛋白。免疫荧光和亚细胞分级分析表明，c-myc标记的克隆c5 fw是不溶性的，主要定位在细胞质中的颗粒的形式。
We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues. several potential phosphorylation sites, and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dug counterpart, We have also isolated another human-related clone c5fw sharing 70% amino their identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5fw protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw). In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gal electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epidermal growth factor thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c-myc-tagged clone c5fw was insoluble and localized mainly in the cytoplasm in the form of granules.