Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p:: Evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth
Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p:: Evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth
复制标题
DOI：
10.1128/mcb.23.17.6187-6199.2003
复制
发表时间：
2003-09-01
影响因子：
5.3
通讯作者：
Maitra, U
中科院分区：
文献类型：
--
作者：
Basu, U;Si, K;Maitra, U
在酿酒酵母中60 S核糖体亚基的合成需要Tif 6p，哺乳动物真核翻译起始因子6（eIF 6）的酵母同源物。在目前的工作中，我们已经从兔网织红细胞裂解物中分离出一种蛋白激酶，该蛋白激酶能够磷酸化重组人eIF 6。质谱分析以及纯化的激酶的抗原特性鉴定其为酪蛋白激酶I。体外磷酸化位点从酵母到哺乳动物都高度保守，被鉴定为位置174（主要位点）和175（次要位点）的丝氨酸残基。同源酵母蛋白Tif 6p也在酵母细胞中被体内磷酸化。Tif 6p在丝氨酸-174处突变为丙氨酸显著降低磷酸化，并导致细胞生长和活力丧失。当Ser-174和Ser-175都突变为丙氨酸时，Tif 6p的磷酸化被完全消除。此外，虽然野生型Tif 6p分布在酵母细胞的细胞核和细胞质中，但突变体Tif 6p（具有Ser 174 Ala和Ser 175 Ala）成为组成型核蛋白。这些结果表明，可磷酸化的Ser-174和Ser-175在Tif 6p的核输出中起关键作用。
The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.