Enhanced interaction between tubulin and microtubule-associated protein 2 via inhibition of MAP kinase and CDC2 kinase by paclitaxel.
Paclitaxel, an anti-mitotic anti-cancer agent, is active against solid tumors. The inhibition of depolymerization and promotion of microtubular assembly are essential for the anti-tumor activity of paclitaxel. Microtubule-associated proteins (MAPs) co-polymerize with tubulin and play some roles in microtubular dynamics. We examined the effect of paclitaxel on the interaction between tubulin and MAPs. Human lung-cancer cells, PC-14, were synchronized to G1/S border by the thymidine-double-block technique. After release from exposure to thymidine, the cells were treated briefly with 2 nM paclitaxel and the levels of alpha and beta tubulins and MAPs were examined after various times. Immunoblot analysis of paclitaxel-treated cells showed no changes in the overall expression of alpha and beta tubulins, microtubule-associated protein 2 (MAP2) or MAPs in comparison with controls. The samples were immunoprecipitated with anti-alpha- and anti-beta-tubulin antibodies and reblotted with an anti-MAP2 antibody, which showed that the amount of co-immuno-precipitated MAP2 in the synchronized cells, were increased by the brief paclitaxel treatment. These results suggest that paclitaxel treatment enhances the interaction between alpha and beta tubulins and MAP2. Since the phosphorylation state of MAP2 regulates the affinity of MAP2 for tubulins, and mitogen-activated protein (MAP) kinase is considered to be one of the kinases responsible for MAP2 phosphorylation, the effect of paclitaxel treatment on the MAP-kinase activity of synchronized PC-14 cells was examined. Two bands with molecular masses of 42 and 44 kDa were detected by an "intra-gel" MAP-kinase assay using myelin basic protein as the substrate. Paclitaxel treatment inhibited the MAP-kinase activity of PC-14 cells and inhibition was maximal at the G2/M phase of the cell cycle. Similar, concentration-dependent inhibition by paclitaxel of cellular MAP kinase of human synchronized small-cell lung carcinoma, H69, was observed. No inhibition of the MAP kinase of the paclitaxel-resistant sub-line H69/Txl by paclitaxel was observed, suggesting that some change of the MAP-kinase cascade had occurred in these cells. No direct inhibition of MAP-kinase activity by paclitaxel was observed in the cell-free assay (in vitro), suggesting that paclitaxel did not inhibit MAP kinase directly. Since it has been speculated that p34cdc2 kinase is also a kinase that phosphorylates MAP2, the effect of paclitaxel treatment on the p34cdc2-kinase activity of synchronized PC-14 and PC-9 cells was examined. Paclitaxel inhibited p34cdc2-kinase activation at the G2/M phase. These results suggest that paclitaxel inhibited MAP kinase and p34cdc2 kinase in vivo indirectly. These actions of paclitaxel may be responsible for the increased affinity between MAP2 and tubulins that it induces.
Nishio
K
K
Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
Arioka
H
H
Ishida
T
T
Fukumoto
H
H
Kurokawa
H
H
Sata
M
M
Ohata
M
M
Saijo
N
N
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000201:enzymology / Q000378:metabolism; D002453:Cell Cycle; D002474:Cell-Free System; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology*; D010766:Phosphorylation; D014404:Tubulin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D017239:Paclitaxel