Mechanism of ribosomal p70S6 kinase activation by granulocyte macrophage colony-stimulating factor in neutrophils -: Cooperation of a MEK-related, THR421/SER424 kinase and a rapamycin-sensitive, mTOR-related THR389 kinase
Mechanism of ribosomal p70S6 kinase activation by granulocyte macrophage colony-stimulating factor in neutrophils -: Cooperation of a MEK-related, THR421/SER424 kinase and a rapamycin-sensitive, mTOR-related THR389 kinase
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DOI：
10.1074/jbc.m300376200
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发表时间：
2003-07-25
影响因子：
4.8
通讯作者：
Gomez-Cambronero, J
中科院分区：
文献类型：
--
作者：
Lehman, JA;Calvo, V;Gomez-Cambronero, J
我们在这里首次报道了在造血细胞、中性粒细胞中检测到核糖体p70S6激酶（p70S6K），并通过粒细胞巨噬细胞集落刺激因子（GM-CSF）刺激其酶活性。GM-CSF修饰了酶的V-max（从7.2到20.5 pmol/min/mg），诱导了p70S6K残基Thr（389）和Thr(421)/Ser（424）的时间和剂量依赖性磷酸化。免疫抑制剂大环内酯类雷帕霉素导致Western blot中磷酸化苏氨酸（389）条带的强度降低，或者磷酸化苏氨酸(421)/丝氨酸（424）条带的相对迁移率降低（与磷酸盐的损失一致），但不是同时发生的。免疫抑制剂FK506未能抑制p70S6K的激活，但能够挽救雷帕霉素诱导的降速，这表明雷帕霉素（mTOR）激酶在哺乳动物靶点中的作用。雷帕霉素对p70S6K的体外酶活性也有抑制作用（IC50为0.2 nM）。但对活性的抑制并不完全，仅为40 - 50%，说明中性粒细胞p70S6K活性具有雷帕霉素耐药成分。在雷帕霉素处理前，用丝裂原活化蛋白激酶（MAPK）激酶（MEK）抑制剂PD-98059对细胞进行预孵育，可以完全抑制该成分。这表明来自MEK/ MAPK途径的激酶也在p70S6K激活中起作用。因此，GM-CSF可导致一种抗雷帕霉素的mapk相关激酶(靶向Thr（421)/ Ser(424) S6K磷酸化）和一种对雷帕霉素敏感的mtor相关激酶（靶向Thr(389)）的双重激活，这两者是实现中性粒细胞p70S6K完全激活所必需的。
We report here for the first time the detection of the ribosomal p70S6 kinase (p70S6K) in a hematopoietic cell, the neutrophil, and the stimulation of its enzymatic activity by granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF modified the V-max of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr(389) and Thr(421)/Ser(424). The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr(389) bands in Western blots, or as a downshift in the relative mobility of phospho-Thr(421)/ Ser(424) bands ( consistent with the loss of phosphate), but not both simultaneously. The immunosuppressant FK506 failed to inhibit p70S6K activation, but was able to rescue the rapamycin-induced downshift, pointing to a role for the mammalian target of rapamycin ( mTOR) kinase. Rapamycin also caused an inhibition (IC50 0.2 nM) of the in vitro enzymatic activity of p70S6K. However, the inhibition of activity was not complete, but only a 40 - 50%, indicating that neutrophil p70S6K activity has a rapamycin-resistant component. This component was totally inhibited by pre-incubating the cells with the mitogen-activated protein kinase ( MAPK) kinase (MEK) inhibitor PD-98059 prior to treatment with rapamycin. This indicated that a kinase from the MEK/ MAPK pathway also plays a role in p70S6K activation. Thus, GM-CSF causes the dual activation of a rapamycin-resistant, MAPK-related kinase, that targets Thr(421)/ Ser(424) S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr(389), both of which are needed in cooperation to achieve full activation of neutrophil p70S6K.