Kleinberger-Doron N, Shelah N, Capone R, Gazit A, Levitzki A
Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.
Exp Cell Res. 1998 Jun 15;241(2):340-51. doi: 10.1006/excr.1998.4061.
We have previously reported that certain tyrphostins which block EGF-R phosphorylation in cell-free systems fail to do so in intact cells. Nevertheless, we found that this family of tyrphostins inhibits both EGF- and calf serum-induced cell growth and DNA synthesis [Osherov, N.A., Gazit, C., Gilon, and Levitzki, A. (1993). Selective inhibition of the EGF and HER2/Neu receptors by Tyrphostins. J. Biol. Chem. 268, 11134-11142.] Now we show that these tyrphostins exert their inhibitory activity even when added at a time when the cells have already passed their restriction point and receptor activation is no longer necessary. AG555 and AG556 arrest 85% of the cells at late G1, whereas AG490 and AG494 cause cells to arrest at late G1 and during S phase. No arrest occurs during G2 or M phase. Further analysis revealed that these tyrphostins act by inhibiting the activation of the enzyme Cdk2 without affecting its levels or its intrinsic kinase activity. Furthermore, they do not alter the association of Cdk2 to cyclin E or cyclin A or to the inhibitory proteins p21 and p27. These compounds also have no effect on the activating phosphorylation of Cdk2 by Cdk2 activating kinase (CAK) and no effect on the catalytic domain of cdc25 phosphatase. These compounds lead to the accumulation of phosphorylated Cdk2 on tyrosine 15 which is most probably the cause for its inhibition leading to cell cycle arrest at G1/S. A structure-activity relationship study defines a very precise pharmacophore, suggesting a unique molecular target not yet identified and which is most probably involved in the regulation of the tyrosine-phosphorylated state of Cdk2. These compounds represent a new class of cell proliferation blockers whose target is Cdk2 activation.
我们之前报道过，某些在无细胞体系中能阻断表皮生长因子受体（EGF-R）磷酸化的 tyrphostin 在完整细胞中却无法做到这一点。然而，我们发现这类 tyrphostin 能抑制 EGF 和小牛血清诱导的细胞生长及 DNA 合成[Osherov, N.A., Gazit, C., Gilon, and Levitzki, A. (1993). Tyrphostins 对 EGF 和 HER2/Neu 受体的选择性抑制。《生物化学杂志》268, 11134 - 11142。]现在我们表明，即使在细胞已经通过其限制点且受体激活不再必要时添加这些 tyrphostin，它们仍能发挥抑制活性。AG555 和 AG556 使 85%的细胞在 G1 晚期停滞，而 AG490 和 AG494 导致细胞在 G1 晚期和 S 期停滞。在 G2 期或 M 期不发生停滞。进一步分析表明，这些 tyrphostin 通过抑制细胞周期蛋白依赖性激酶 2（Cdk2）的激活起作用，而不影响其水平或内在激酶活性。此外，它们不会改变 Cdk2 与细胞周期蛋白 E 或细胞周期蛋白 A 或抑制性蛋白 p21 和 p27 的结合。这些化合物对 Cdk2 激活激酶（CAK）介导的 Cdk2 激活磷酸化也没有影响，对细胞分裂周期蛋白 磷酸酶（cdc25 phosphatase）的催化结构域也没有影响。这些化合物导致酪氨酸 15 位点磷酸化的 Cdk2 积累，这很可能是其抑制作用的原因，导致细胞周期在 G1/S 期停滞。一项构效关系研究确定了一个非常精确的药效基团，表明存在一个尚未确定的独特分子靶点，很可能参与调节 Cdk2 的酪氨酸磷酸化状态。这些化合物代表了一类新的细胞增殖阻滞剂，其靶点是 Cdk2 的激活。
