Phosphorylation sites in the autoinhibitory domain participate in p70s6k activation loop phosphorylation
Phosphorylation sites in the autoinhibitory domain participate in p70s6k activation loop phosphorylation
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DOI：
10.1074/jbc.273.24.14845
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发表时间：
1998-06-12
影响因子：
4.8
通讯作者：
Thomas, G
中科院分区：
文献类型：
--
作者：
Dennis, PB;Pullen, N;Thomas, G
在这里，我们使用p70(S6K)截断和点突变来阐明羧基末端自抑制结构域P磷酸化位点在激酶激活中所起的作用。早期的研究表明，p70(S6K)氨基末端的截断严重损害了激酶的激活，但这种作用可以通过删除羧基末端而逆转，这同时导致激活环中Thr(229)磷酸化的解除调节(Dennis，P.B.，Pullen，N.，Kozma，S.C.和Thomas，G.(1996)Mol)。牢房。比奥尔。16、6242-6251)。在本研究中，用酸性残基取代四个自抑制结构域P位点，很大程度上模拟了羧基末端的缺失，主要是通过挽救氨基末端截断引起的激酶激活来实现的。然而，这些突变不会解除对Thr(229)磷酸化的调控，这表明在完整的激酶中参与了另一种调控元件。这个元件似乎是Thr(389)磷酸化，因为在包含P突变的p70(S6K)变异体中这个位置的酸性残基的替换导致基础Thr(229)磷酸化和激酶活性的大幅增加。相反，Thr(389)处的丙氨酸取代阻止了这两种反应。与这些数据一致，我们表明，含有酸性P和Thr(389)取代的突变体是新发现的Thr(229)激酶，磷酸肌醇依赖的激酶-1(Pullen，N.，Dennis，P.B.，Andjelkovic，M.，Dufner，A.，Kozma，S.，Hemmings，B.A.和Thomas，G.(1998)Science 279,707-710)的优秀体外底物，而磷酸肌醇依赖的激酶-L不能很好地利用只有一组突变的两个p70(S6K)变体。这些发现表明，P位的磷酸化与Thr(389)的磷酸化一起，通过空间内机制控制Thr(229)的磷酸化。
Here we have employed p70(s6k) truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain P phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70(s6k) amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr(229) phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain P sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr(229) phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr(389) phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the P mutations leads to a large increase in basal Thr(229) phosphorylation and kinase activity. In contrast, an alanine substitution at Thr(389) blocks both responses. Consistent with these data, we, show that a mutant harboring the acidic P and Thr(389) substitutions is an excellent in vitro substrate for the newly identified Thr(229) kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-l poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the P sites, in cooperation with Thr(389) phosphorylation, controls Thr(229) phosphorylation through an intrasteric mechanism.