The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases
The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases
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DOI：
10.1128/mcb.16.11.6242
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发表时间：
1996-11
影响因子：
5.3
通讯作者：
P. Dennis;Nicholas Pullen;S. Kozma;George Thomas
中科院分区：
文献类型：
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作者：
P. Dennis;Nicholas Pullen;S. Kozma;George Thomas
有丝分裂原诱导的p70(S6K)活化与特定位点的磷酸化有关，这些位点受到免疫抑制剂雷帕霉素、真菌代谢物Wortmannin和甲基黄嘌呤SQ20006的负面影响。最近的报道集中在P85(S6K)亚型的氨基末端在调节激酶活性中的作用，观察到氨基末端截断突变体在雷帕霉素存在的情况下被激活，同时保持其对Wortmannin的敏感性。在这里，我们表明，前面描述的氨基和羧基末端截断对激酶活性的影响最终反映在酶的磷酸化状态中。将主要的雷帕霉素靶向磷酸化位点T-389突变为酸性残基，会产生一种对Wortmannin或SQ20006的抗性与对雷帕霉素一样的激酶形式，这与之前的观察结果一致，即T-389是所有三种抑制剂的共同靶点。氨基酸末端前54个残基的截断阻断了血清诱导的三个雷帕霉素敏感部位的磷酸化，T-229位于激活环，T-389和S-404位于连接区。这与血清激活该激酶的能力严重降低有关。然而，由于去掉氨基末端而导致的丝裂原活性的丧失可以通过额外截断羧基末端结构域来逆转，所产生的突变体显示剩余的两个雷帕霉素敏感位点T-229和T-389被磷酸化。在这个双截断突变体中，T-229的磷酸化发生在基础状态，而需要有丝分裂原刺激才能诱导T-389磷酸化的急剧上调。在雷帕霉素存在的情况下，这两个位点的磷酸化不会受到影响，这表明负责这些位点磷酸化的激酶不会被大环内酯类化合物抑制。相反，在Wortmannin或SQ20006存在下，双截断突变体的激活被阻断，这些药物完全阻止T-389的磷酸化，而对T-229的磷酸化只有轻微的影响。当T-389位在双截断背景中突变为酸性残基时，所产生的突变体的激活对Wortmannin和SQ20006块不敏感，但有趣的是，在雷帕霉素存在的情况下，突变体的激活程度显著高于对照。这些数据与T-389是主要调节磷酸化位点的假设一致，该位点与自抑制结构域S/TP位点的过度磷酸化结合在一起，受到外部效应物的强烈调控，而T-229的磷酸化主要受内部机制的调节。
Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.