Phosphoenolpyruvate-dependent tubulin-pyruvate kinase interaction at different organizational levels.
Evidence for the direct binding of pyruvate kinase to tubulin/microtubule and for the inhibitory effect of phosphoenolpyruvate on tubulin-enzyme hetero-association were provided by surface plasmon resonance and pelleting experiments. Electron microscopy revealed that pyruvate kinase induces depolymerization of paclitaxel-stabilized microtubules into large oligomeric aggregates and bundles the tubules in a salt concentration-dependent manner. The C-terminal "tail"-free microtubules did not bind pyruvate kinase, suggesting the crucial role of the C-terminal segments in the binding of kinase. Immunoblotting and polymerization experiments with cell-free brain extract revealed that pyruvate kinase specifically binds to microtubules, the binding of pyruvate kinase impedes microtubule assembly, and phosphoenolpyruvate counteracts the destabilization of microtubules induced by pyruvate kinase. We also showed by immunostaining the juxtanuclear localization of pyruvate kinase in intact L929 cells and that this localization was influenced by treatments with paclitaxel or vinblastine. These findings suggest that the distribution of the enzyme may be controlled by the microtubular network in vivo.
Kovacs
Janos
J
Department of General Zoology, Eötvös Loránd University, P.O. Box 330, H-1445 Budapest, Hungary.
Low
Peter
P
Pacz
Anita
A
Horvath
Istvan
I
Olah
Judit
J
Ovadi
Judit
J
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*; D014747:Vinblastine / Q000494:pharmacology
D000972:Antineoplastic Agents, Phytogenic; D014404:Tubulin; D014747:Vinblastine; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D017239:Paclitaxel