Alessi D R, Cuenda A, Cohen P, Dudley D T, Saltiel A R
Department of Biochemistry, The University, Dundee.
J Biol Chem. 1995 Nov 17;270(46):27489-94. doi: 10.1074/jbc.270.46.27489.
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 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) 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 kinase 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 microM) inhibited the activation of p42MAPK 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.
先前的研究表明，PD 098059能够抑制丝裂原活化蛋白激酶激酶-1（MAPKK1）的去磷酸化形式以及一种组成型活性较低的突变型MAPKK1（S217E，S221E）（达德利，D.T.，庞，L.，德克尔，S.J.，布里奇斯，A.J.，以及萨尔蒂尔，A.R.（1995年）《美国国家科学院院刊》92，7686 - 7689）。在此我们报告，PD 098059并不抑制Raf激活的MAPKK1，而是在体外能够以与抑制去磷酸化MAPKK1或MAPKK1（S217E，S221E）相似的浓度（IC50 = 2 - 7 microM）阻止Raf或MEK激酶对MAPKK1的激活。PD 098059以高得多的IC50值（50 microM）抑制Raf对MAPKK2的激活，并且不抑制其他Raf或MEK激酶底物的磷酸化，这表明它通过与MAPKK1的无活性形式结合来发挥作用。PD 098059在瑞士3T3细胞中也作为MAPKK激活的特异性抑制剂，抑制多种激动剂对其80 - 90%的激活。PD 098059在体外和体内的高度特异性表现为：在体外它不抑制18种蛋白丝氨酸/苏氨酸激酶（包括另外两种MAPKK同源物）；在体内它不抑制参与KB和PC12细胞中应激和白细胞介素-1刺激的激酶级联反应的MAPKK和MAP激酶同源物的激活；在瑞士3T3细胞中它不抑制胰岛素或表皮生长因子对p70 S6激酶的激活。PD 098059（50 microM）抑制瑞士3T3细胞中p42MAPK和MAP激酶激活的蛋白激酶-1亚型的激活，但抑制程度取决于任何特定激动剂对c - Raf和MAPKK的激活强度，并证明了该激酶级联反应巨大的放大潜力。PD 098059不仅在瑞士3T3细胞中不抑制血小板衍生生长因子、血清、胰岛素和佛波酯对Raf的激活，实际上还增强了Raf活性。血小板衍生生长因子对Raf的激活速率增加了3倍，并且阻止了10分钟后发生的后续失活。这些结果表明，Raf的激活受到抑制，并且其失活被MAP激酶途径的下游组分加速。
