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

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 aggregatesandbundlesthetubulesinasaltconcentrationdependent 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. Microtubules (MT)1 constitute a crucial part of the cytoskeleton and are involved in a variety of cell functions such as maintenance of shape, organization of intracellular transport, motility, and cell division. The polymerization dynamics of MT is under strict control (1). In addition to small molecules (inorganic ions, guanine nucleotides, and drugs), numerous proteins are known to interact with MT as positive regulators of MT assembly (MAPs) either by promoting the polymerization of tubulin or by stabilizing MT (1, 2). Several other proteins including Op18/stathmin, katanin, and some kinesin-like proteins act as destabilizers (1, 2).As shown by various methods including co-sedimentation assays and immunostaining, many glycolytic enzymes can interact transiently with the actin and/or microtubular network either in vitro or in vivo; the properties of associated partners (e.g. the stability of cytoskeleton and the activity of the enzyme) are deeply influenced by the mutual interactions (Refs. 3-5; for review see Ref. 6). Recently, 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).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 PK with MTs in brain extract, the protein composition of which approximates that of the living cells. We present evidence on the crucial role of acidic Cterminal fragments of MTs in the PK binding. We show the modulating role of phosphoenolpyruvate (PEP) on tubulin-PK as well as on MT-PK interaction at di...

The Invs Gene Encodes a Microtubule-Associated Protein

“…Precipitated proteins were separated by SDS-PAGE followed by immunoblotting. Co-pelleting assays were performed as described previously (28). Briefly, extracts from MDCK cells treated with colcemid or paclitaxel were centrifuged at 100.000 ϫ g for 20 min (Rotor RP55-S; M120 Microultracentrifuge, Sorvall, Kendro Laboratory Products GmbH, Langenselbold, Germany).…”

Fueled by microtubules: Does tubulin dimer/polymer partitioning regulate intracellular metabolism?

“…In support of this hypothesis, several glycolytic enzymes are known to regulate MT assembly. For example, PK destabilizes MTs and impedes MT assembly [Vertessy et al, 1999;Kovacs et al, 2003]. PFK from Dictystelium discoideum is also an inhibitor of MT polymerization [Orosz et al, 1999].…”

“…2. For phosphoglucose isomerase [Walsh et al, 1989], aldolase [Walsh et al, 1989;Volker et al, 1995], triosephosphate isomerase (TPI) [Orosz et al, 2000], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [Walsh et al, 1989;Somers et al, 1990;Volker et al, 1995;Andrade et al, 2004;Chuong et al, 2004], phosphoglycerate kinase (PGK) [Walsh et al, 1989], and pyruvate kinase (PK) [Walsh et al, 1989;Volker et al, 1995;Kovacs et al, 2003], binding has been demonstrated by copelleting with MTs. In the case of TPI, binding of the native protein is relatively weak, but a mutant form found in a human disease shows much greater to binding to MTs [Orosz et al, 1999].…”

“…Others, including hexokinase (HK), phosphofructokinase (PFK), GAPDH, enolase, and PK, have been shown to colocalize with MTs in brain extracts (HK, Wagner et al [2001]; PK, Kovacs et al [2003]), myoblasts/muscle (enolase [Keller et al, 2007]), BHK cells (GAPDH [Andrade et al, 2004]) or melanoma cells (PFK [Glass-Marmor and Beitner, 1999]). Several enzymes bind both tubulin dimers and MT polymers, including HK [Wagner et al, 2001], enolase [Keller, 2007], and PK [Kovacs et al, 2003]. Most of the above observations Metabolites cross the outer mitochondrial membrane through VDAC (purple channels).…”

Functional Taxonomy of Bacterial Hyperstructures

“…The tubulin cytoskeleton has also been proposed as being intimately related to metabolic activity. Glyceraldehyde-3-phosphate dehydrogenase can interact with microtubules to bring them together (104,243,270); triosephosphate isomerase, hexokinase, phosphofructokinase, pyruvate kinase, and aldolase interact with microtubular proteins and a hyperstructure can exist in which enzyme properties and fluxes are altered (210); and pyruvate kinase hinders microtubule assembly in vitro unless phosphoenolpyruvate is present (124).…”