Pyruvate Kinase as a Microtubule Destabilizing Factor in Vitro Bea ´ta G. Ve ´rtessy,* ,1 Do ´ra Ba ´ nkfalvi,* Ja ´ nos Kova ´cs,† Pe ´ter Lo ¨w,† Attila Lehotzky,‡ and Judit Ova ´ di* *Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, POB 7, H-1518, Budapest, Hungary; †Department of General Zoology, University of Eo ¨tvo ¨s Lora ´ nd, Budapest, Hungary; and ‡Pharmaceutical Works of Gedeon Richter, Budapest, Hungary Received December 3, 1998 Endogenous control of microtubule dynamism is es- sential in many cell types. Numerous microtubule- adhering proteins stabilize the polymer status, while very few protein factors are described with opposite effects. The brain- and muscle-specific M1 isoform of the enzyme pyruvate kinase is investigated here in this respect. Three pieces of evidence indicate antimi- crotubular effects of this protein. (1) Pyruvate kinase inhibits taxol-induced tubulin polymerization into mi- crotubules as revealed by turbidimetry. (2) Pelleting experiments show that pyruvate kinase partially dis- assembles taxol-stabilized microtubules into less sedi- mentable oligomers leading to the appearance of tu- bulin in the supernatant fractions. (3) Electron microscopy reveals the kinase-induced formation of great amounts of thread-like tubulin oligomers which tend to accumulate in a light/less sedimentable frac- tion. Immunoelectron micrographs using labeled anti- body against pyruvate kinase provide evidence for the binding of pyruvate kinase to the thread-like oligo- meric forms. The present data allow the assumption that pyruvate kinase may display multiple regulatory functions as a glycolytic control enzyme and as a mod- ulator of microtubule dynamism. © 1999 Academic Press Key Words: microtubule antagonism; pyruvate ki- nase; microtubule dynamism; turbidimetry; differen- tial pelleting; microtubule ultrastructure. Microtubules (MTs), polymers of the - tubulin het- erodimer, are central to numerous physiological phe- nomena, e.g., intracellular transport, cell shape forma- tion and mitosis (1). The dynamic nature of the tubulin-MT equilibrium is indispensable for normal cell function as for example in neuronal plasticity and mitotic spindle formation/disassembly (2, 3, and refer- ences therein). The nondividing neurons of the adult mammalian brain require effective control of tubulin/ MT dynamism especially in the process of neurite ex- tension (4, 5). Endogenous factors responsible for reg- ulation of MT dynamism are expressed in a tissue- and cell cycle-specific manner so that the physiological sta- tus of the cell is imprinted in the pattern of expression of the regulatory factors (6). The numerous MT- associated proteins (MAPs) stimulate MT assembly by preferentially binding to and stabilizing the tubules (1). MAP-MT interactions are usually regulated by MAP phosphorylation (7). Lack of the MAP-stabilizing effects indirectly enhances MT disassembly, however, recently some proteins were identified at the molecular level which directly act in an antimicrotubular way (8 –10). One of these factors is stathmin/op18, a small phosphoprotein present in high amounts in different tumour cells (11), while other factors belong to the kinesin family. These factors seem to employ different antagonistic mechanisms of action (10, 12, 13). Contin- ued search for endogenous regulators of MT dynamism is urged by the essential need for understanding the tubulin assembly/disassembly process under different physiological circumstances and in different tissues. Several enzymes of glycolysis, indispensable in brain as the first step in the catabolism of the most important neuronal energy source glucose, are present at concen- trations significantly exceeding the requirement as simple catalysts (14). In vivo evidence from axonal transport indicates at least transient proximity of gly- colytic enzymes and MTs (15, 16). In vitro studies doc- umented significant binding of some enzymes (e.g., phosphofructokinase, glyceraldehyde-3-phosphate de- hydrogenase, aldolase, pyruvate kinase (PK)) to tubu- lin and MTs (17, 18). Enzyme binding modulated mi- crotubular ultrastructure in the case of glycer- aldehyde-3-phosphate dehydrogenase (19, 20) and 1 To whom correspondence should be addressed. Fax: +361 4 665465. E-mail: vertessy@enzim.hu. Abbreviations used: MT, microtubule; MAP, microtubule- associated protein; PK, pyruvate kinase; SDS/PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Biochemical and Biophysical Research Communications 254, 430 – 435 (1999) Article ID bbrc.1998.9957, available online at http://www.idealibrary.com on 430 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.