PD-098059 IS A SPECIFIC INHIBITOR OF THE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE KINASE IN-VITRO AND IN-VIVO
PD-098059 IS A SPECIFIC INHIBITOR OF THE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE KINASE IN-VITRO AND IN-VIVO
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DOI：
10.1074/jbc.270.46.27489
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发表时间：
1995-11-17
影响因子：
4.8
通讯作者：
SALTIEL, AR
中科院分区：
文献类型：
--
作者：
ALESSI, DR;CUENDA, A;SALTIEL, AR
PD 098059先前已被证明可以抑制有丝分裂原活化蛋白激酶1（MAPKK1）的去磷酸化形式和突变体MAPKK1(S217E,S221E)，这具有低水平的组成活性(Dudley， D. T., Pang， L., Decker， S. J., Bridges, a . J., and Saltiel, a . R. (1995) Proc. Natl。学会科学。在这里，我们报道PD 098059不抑制Raf激活的MAPKK1，但它在体外阻止Raf或MEK激酶对MAPKK1的激活，浓度（IC50 = 2-7 μ M）与抑制去磷酸化MAPK1或MAPKK1的浓度（S217E,S221E）相似。PD 098059以更高的IC50值（50 μ M）抑制Raf对MAPKK2的激活，并没有抑制其他Raf或MEK激酶底物的磷酸化，表明其通过结合MAPKK1的失活形式发挥作用。PD 098059也是Swiss 3T3细胞中MAPKK活化的特异性抑制剂，可抑制多种激动剂对MAPKK活化的80-90%。098059年PD高度特异性的体外和体内表示未能抑制18对丝氨酸/苏氨酸蛋白激酶(包括其他两个MAPKK同系物)由其未能抑制体内体外激活MAPKK和MAP激酶同系物参与压力和interleukin-1-stimulated ki nase瀑布KB和PC12细胞,激活的和缺乏抑制p70 S6激酶通过胰岛素或表皮生长因子在瑞士3 t3细胞。PD 098059 （50 μ M）抑制了瑞士3T3细胞中p42（MAPK）和MAP激酶活化蛋白激酶-1同型的激活，但抑制程度取决于c-Raf和MAPKK被任何特定激动剂激活的效力，并证明了这种激酶级联的巨大扩增潜力，PD 098059不仅未能抑制血小板衍生生长因子、血清、胰岛素、血小板衍生生长因子激活Raf的速率增加了3倍，随后10分钟后发生的失活被阻止，这些结果表明，Raf的激活受到抑制，其失活被MAP激酶途径的下游组分加速。
PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7686-7689), Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 mu M) similar to those concentrations that inhibit dephosphorylated MAPK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 mu M) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists, The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated ki nase cascades in KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 mu M) inhibited the activation of p42(MAPK) and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade, PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity, The rate of activation of Raf by platelet derived growth factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented, These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway.