KT5720 and U-98017 inhibit MAPK and alter the cytoskeleton and cell morphology
KT5720 and U-98017 inhibit MAPK and alter the cytoskeleton and cell morphology
复制标题
DOI：
10.1002/(sici)1097-4652(199809)176:3
复制
发表时间：
1998-09-01
影响因子：
5.6
通讯作者：
Abraham, I
中科院分区：
文献类型：
--
作者：
Olsen, MK;Reszka, AA;Abraham, I
我们先前在中国仓鼠卵巢（CHO）紫杉醇依赖性细胞筛选紫杉醇模拟物中鉴定了KT 5720和U-98017作为具有紫杉醇（紫杉醇）样活性的药物。纯化的脑微管蛋白的体外聚合基本上不受这些化合物的影响，这表明，与紫杉醇不同，这些药物不直接影响微管蛋白。然而，这些化合物引起完整细胞中细胞骨架的深刻重排，包括微管长度的明显改变、细胞的重叠和细胞大小的增加。我们表明，KT 5720和U-98017有效地抑制丝裂原活化蛋白激酶（MAPK）的活性在体外。Staurosporine是一种弱的MAPK抑制剂，但却是cAMP依赖性蛋白激酶A（PKA）活性、磷脂/Ca++依赖性激酶（PKC）和cdc 2的强效抑制剂，不会引起类似的变化。此外，在U-98017中生长的紫杉醇依赖性细胞具有显著降低的刺激MAPK水平。与这些结果相关，我们已经证实了MAPK在细胞中的分离的微管蛋白和微管中的存在。我们已经研究了这一假设，即这些化合物是通过抑制MAPK的作用，通过抑制微管相关蛋白的磷酸化改变微管。在CHO细胞中转染的MAPKK显性负突变抑制MAPK的活化。携带该显性突变体的转染子具有受损的MAPK活化和改变的细胞形态，在某些方面类似于用KT 5720和U-98017观察到的。这些结果支持MAPK家族成员在微管动力学控制中的作用，并表明在完整细胞中U-98017和KT 5720通过抑制激酶如MAPK来实现改变细胞骨架和支持紫杉醇依赖性细胞的部分生长的作用。J.细胞。176：525-536，1998。(C)1998 Wiley-Liss，Inc.
We previously identified KT5720 and U-98017 as agents that had paclitaxel (taxol)-like activity in a Chinese hamster ovary (CHO) paclitaxel-dependent cell screen for paclitaxel mimetics. In vitro polymerization of purified brain tubulin is not affected substantially by these compounds, suggesting that, unlike paclitaxel, these agents do not directly affect tubulin. However, these compounds cause profound rearrangements of the cytoskeleton in intact cells, including an apparent alteration of microtubule length, overlapping of cells, and an increase in cell size. We show that KT5720 and U-98017 effectively inhibit mitogen-activated protein kinase (MAPK) activity in vitro. Staurosporine, a poor inhibitor of MAPK but a potent inhibitor of cAMP-dependent protein kinase A (PKA) activity, phospholipid/Ca++-dependent kinase (PKC), and cdc2, does not cause similar changes. In addition, paclitaxel-dependent cells grown in U-98017 have substantially decreased levels of stimulated MAPK. In correlation with these results, we have confirmed the presence of MAPK in isolated tubulin and microtubules in cells. We have examined the hypothesis that these compounds are working through inhibition of MAPK to alter microtubules by inhibiting the phosphorylation of microtubule-associated proteins. A MAPKK dominant negative mutation transfected in CHO cells inhibits activation of MAPK. Transfectants carrying this dominant mutant have impaired activation of MAPK and an altered cell morphology, similar in some respects to that seen with KT5720 and U-98017. These results support a role for MAPK family members in the control of microtubule dynamics and suggest that in intact cells U-98017 and KT5720 achieve their effects of altering cytoskeleton and supporting partial growth of paclitaxel-dependent cells through inhibition of kinases such as MAPK. J. Cell. Physiol. 176:525-536, 1998. (C) 1998 Wiley-Liss, Inc.