Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1
Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1
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DOI：
10.1159/000104169
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Wenger, Roland H.
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文献类型：
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作者：
Eckhardt, Katrin;Troeger, Juliane;Wenger, Roland H.
帕斯金将酵母中的能量流和蛋白质合成联系在一起，调节哺乳动物的糖原合成，并参与葡萄糖刺激的胰岛β细胞胰岛素的产生。利用新产生的单抗，帕斯金被定位在人类睾丸生殖细胞的细胞核和人类精子尾巴的中段。在HeLa细胞中，内源性Paskin除了胞质定位外，还观察到了斑点状的核图案。通过酵母双杂交筛选，我们确定多功能真核翻译延伸因子eEF1A1是Paskin的一个新的相互作用伙伴。通过哺乳动物双杂交和GST下拉分析，这种相互作用被映射到Paskin的Pas A和Kinase结构域以及eEF1A1的C末端。激酶分析、质谱分析和定点突变显示，Paskin自动磷酸化和eEF1A1靶向磷酸化主要但不限于Thr432。野生型但不是激酶失活的帕斯金增加了报告基因cRNA的体外翻译。虽然eEF1A1不定位于细胞核，但它与Paskin共同定位于HeLa细胞的细胞质。这两种蛋白质在精子尾部中部的定位也非常相似。这些数据表明，在体细胞和精子细胞中，eEF1A1通过帕斯金依赖的磷酸化来调节。版权所有(C)2007 S.Karger AG，巴塞尔。
PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells. Copyright (c) 2007 S. Karger AG, Basel.