KT5720 AND U-98017 INHIBIT MAPK AND ALTER THE CYTOSKELETON AND CELL MORPHOLOGY

We previously identified KT5720 and U-98017 as agents that had paclita xel (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 comp ounds, suggesting that, unlike paclitaxel, these agents do not directl y affect tubulin. However, these compounds cause profound rearrangemen ts of the cytoskeleton in intact cells, including an apparent alterati on of microtubule length, overlapping oi cells, and an increase in cel l size. We show that KT5720 and U-98017 effectively inhibit mitogen-ac tivated protein kinase (MAPK) activity in vitro. Staurosporine, a poor inhibitor of MAPK but a potent inhibitor of cAMP-dependent protein ki nase A (PKA) activity, phospholipid/Ca++-dependent kinase (PKC), and c dc2, does not cause similar changes. In addition, paclitaxel-dependent cells grown in U-98017 have substantially decreased levels of stimula ted MAPK. In correlation with these results, we have confirmed the pre sence of MAPK in isolated tubulin and microtubules in cells. We have e xamined the hypothesis that these compounds are working through inhibi tion of MAPK to alter microtubules by inhibiting the phosphorylation o f microtubule-associated proteins. A MAPKK dominant negative mutation transfected in CHO cells inhibits activation of MAPK. Transfectants ca rrying this dominant mutant have impaired activation of MAPK and an al tered cell morphology, similar in some respects to that seen with KT57 20 and U-98017. These results support a role for MAPK family members i n 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 inhib ition of kinases such as MAPK. J. Cell. Physiol. 176:525-536, 1998. (C ) 1998 Wiley-Liss, Inc.