Phosphoenolpyruvate-dependent Tubulin-Pyruvate Kinase Interaction at Different Organizational Levels

János Kovács,Péter Löw,Anita Pácz,István Horváth,Judit Oláh,Judit Ovádi

doi:10.1074/jbc.m210244200

János Kovács, Péter Löw + Show 4 more

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https://doi.org/10.1074/jbc.m210244200

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Abstract

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.

Highlights

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
In the present study we have further characterized the PKtubulin/MT interaction with purified proteins using surface plasmon resonance technology, and we present data on the heterologous association of pyruvate kinase (PK) with MTs in brain extract, the protein composition of which approximates that of the living cells
We have investigated the effect of ionic strength on the binding of PK to MTs. 2 ␮M PK was added to paclitaxel-stabilized MTs in the presence of either 50 or 150 mM KCl in the polymerizing buffer, and the samples were centrifuged at 100,000 ϫ g, and the pellets were analyzed by transmission electron microscopy

Summary

Introduction

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. We 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. We found that two of the glycolytic enzymes, the M1 isoform of pyruvate kinase (PK) and Dictyostelium discoideum phosphofructokinase, can act as microtubule-destabilizing factors by inhibiting paclitaxel-induced polymerization of tubulin and by promoting disassembly of microtubules into thread-like oligomers [7, 8]. We show the modulating role of phosphoenolpyruvate (PEP) on tubulin-PK as well as on MT-PK interaction at different organization levels and demonstrate that the distribution of PK in mouse fibroblasts is highly dependent on the integrity of microtubular network

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