Missense mutations at the FKBP12-rapamycin-binding site of TOR1
Missense mutations at the FKBP12-rapamycin-binding site of TOR1
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DOI：
10.1016/0378-1119(96)00168-0
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发表时间：
1996-06-12
期刊：
影响因子：
3.5
通讯作者：
Livi, GP
中科院分区：
文献类型：
--
作者：
Freeman, K;Livi, GP
TOR基因首先在酿酒酵母中通过分离对免疫抑制和抗真菌药物雷帕霉素（Rm）表现出显性抗性的突变体来鉴定。最初表征的Rm抗性（Rm（R））TOR-1和TOR 2 -1等位基因含有Arg代替保守的Ser残基，其位于TOR的磷脂酰肌醇（PI）激酶相关结构域附近（TOR-1中的Ser（1972）; TOR 2中的Ser（1975））。随后分离出另外的含有Lys、Ile或Asn取代的自发Rm（R）突变体。由于该Ser是蛋白激酶C磷酸化的潜在位点，我们感兴趣的是确定观察到的Rm（R）是否是由于FKBP 12-Rm-TOR相互作用的空间位阻，或者是否需要在该位点的磷酸化来介导相互作用。使用定点突变，我们用保守残基Ala、替代的潜在磷酸化位点Thr或Asp替换TOR 1的Ser（1972）残基以模拟磷酸化。TOR 1（S1972 A）突变蛋白保留Rm敏感性（Rm（S）），而Thr和Asp取代均赋予Rm（R）。在双杂交试验中，Rm（S）与FKBP 12-Rm相互作用的能力相关：野生型TOR 1和S1972 A突变体都保留了与FKBP 12-Rm相互作用的能力，而S1972 T、S1972 D和S1972 R突变体则不能相互作用。所有的突变体TOR 1蛋白都能够补充tor 1无效等位基因的生长缺陷，这表明Ser（1972）残基可能不是TOR 1在循环细胞中发挥功能所必需的。由于TOR 1（S1972 A）突变蛋白赋予Rm（S）表型，在双杂交试验中与FKBP 12-Rm相互作用，并在体内发挥功能，因此我们得出结论，TOR 1和FKBP 12-Rm之间的相互作用不需要Ser（1972）磷酸化。
The TOR genes were first identified in Saccharomyces cerevisiae by the isolation of mutants which exhibit dominant resistance to the immunosuppressive and antifungal drug rapamycin (Rm). The originally characterized Rm-resistant (Rm(R)) TOR-1 and TOR2-1 alleles contain an Arg in place of a conserved Ser residue, which lies adjacent to the phosphatidylinositol (PI) kinase-related domain of TOR (Ser(1972) in TOR-1; Ser(1975) in TOR2). Additional spontaneous Rm(R) mutants containing Lys, Ile or Asn substitutions were subsequently isolated. As this Ser is a potential site for protein kinase C phosphorylation, we were interested in determining whether the observed Rm(R) is due to steric hindrance of the FKBP12-Rm-TOR interaction or whether phosphorylation at this site is required to mediate the interaction. Using site-directed mutagenesis, we replaced the Ser(1972) residue of TOR1 with either a conservative residue, Ala, an alternative potential phosphorylation site, Thr, or Asp to mimic phosphorylation. The TOR1(S1972A) mutant protein retained Rm sensitivity (Rm(S)), whereas both the Thr and Asp substitutions conferred Rm(R). Rm(S) correlated with the ability to interact with FKBP12-Rm in a two-hybrid assay: both wild-type TOR1 and the S1972A mutant retained the ability to interact with FKBP12-Rm, whereas the S1972T, S1972D and S1972R mutants failed to interact. All mutant TOR1 proteins were able to complement the growth defect of tor1 null alleles, suggesting that the Ser(1972) residue may not be required for TOR1 function in cycling cells. Since a TOR1(S1972A) mutant protein confers a Rm(S) phenotype, interacts with FKBP12-Rm in a two-hybrid assay, and functions in vivo, we conclude that phosphorylation at Ser(1972) is not necessary for the interaction between TOR1 and FKBP12-Rm.