IDENTIFICATION OF AN 11-KDA FKBP12-RAPAMYCIN-BINDING DOMAIN WITHIN THE 289-KDA FKBP12-RAPAMYCIN-ASSOCIATED PROTEIN AND CHARACTERIZATION OF A CRITICAL SERINE RESIDUE
IDENTIFICATION OF AN 11-KDA FKBP12-RAPAMYCIN-BINDING DOMAIN WITHIN THE 289-KDA FKBP12-RAPAMYCIN-ASSOCIATED PROTEIN AND CHARACTERIZATION OF A CRITICAL SERINE RESIDUE
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DOI：
10.1073/pnas.92.11.4947
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发表时间：
1995-05-23
影响因子：
11.1
通讯作者：
SCHREIBER, SL
中科院分区：
文献类型：
--
作者：
CHEN, J;ZHENG, XF;SCHREIBER, SL
天然产物雷帕霉素与其胞内受体 FKBP12 复合，可抑制多种哺乳动物细胞系和酿酒酵母中细胞周期的 G(1) 进程。此前，已鉴定出一种与 FKBP12-雷帕霉素直接相关的哺乳动物蛋白，并且已从人类（指定为 FRAP）[Brown, E. J.、Albers, M. W.、Shin, T. B.、Ichikawa, K.、Keith, C. T.、Lane, W. S. 和 Schreiber, S.L. 中克隆了其编码基因。 (1994) Nature (London) 369, 756-758] 和大鼠（称为 RAFT）[Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. 和 Snyder, S. H. (1994) Cell 78, 35-43]。全长 FRAP 是一种 289 kDa 的蛋白质，包含假定的磷脂酰肌醇激酶结构域。使用体外转录/翻译测定方法结合蛋白水解研究，我们在 FRAP 中鉴定了一个 11-kDa FKBP12-雷帕霉素结合域。该最小结合结构域位于激酶结构域的 N 端，跨越残基 2025-2114。此外，我们还进行了诱变研究来研究 Ser(2035) 的作用，Ser(2035) 是该结构域内蛋白激酶 C 的潜在磷酸化位点。我们现在表明，与野生型蛋白相比，FRAP Ser(2035) --> Ala 突变体显示出相似的结合亲和力，而该位点的所有其他突变（包括磷酸丝氨酸的模拟物）都消除了结合，可能是由于不利的空间相互作用或诱导的构象变化。
Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G(1) progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae. Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) [Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S. and Schreiber, S.L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. and Snyder, S. H. (1994) Cell 78, 35-43]. The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain. Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP. This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114. In addition, we have carried out mutagenesis studies to investigate the role of Ser(2035), a potential phosphorylation site for protein kinase C within this domain. We now show that the FRAP Ser(2035) --> Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes.