Phosphofructokinase from <i>Dictyostelium discoideum</i> Is a Potent Inhibitor of Tubulin Polymerization

Orosz, Ferenc; Santamaría, Belén; Ovádi, Judit; Aragón, J. L.

doi:10.1021/bi981350p

Cited by 14 publications

(9 citation statements)

References 51 publications

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“…Only a few proteins are known to act as destabilizers, such as Op18͞stathmin, katanin, and some kinesin-like proteins (1,2). We have reported recently that the M1 isoform of pyruvate kinase and Dictyostelium discoideum phosphofructokinase inhibit tubulin polymerization or promote disassembly of the MTs to thread-like oligomers in vitro (3)(4)(5).…”

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confidence: 99%

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation

Tirián

Hlavanda

Oláh

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

116

136

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Recently, we isolated from bovine brain a protein, TPPP͞p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via BLAST. Here, we characterized the binding of TPPP͞p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 ؋ 10 4 M ؊1 ⅐s ؊1 ; koff, 5.4 ؋ 10 ؊3 s ؊1 ; and Kd, 2.3 ؋ 10 ؊7 M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP͞p25 into cleavage Drosophila embryos expressing tubulin-GFP fusion protein reveals that TPPP͞p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP͞p25 both in vitro and in vivo.T he cytoplasm of eukaryotic cells contains an elaborate network of cytoskeletal elements, consisting of actin and intermediate filaments and microtubules (MTs) engaged in a variety of cell functions, such as the extension and guidance of neurons or the formation of mitotic spindles required for chromosomal segregation. The polymerization dynamics of tubulin to MTs is under strict control (1). Numerous proteins have been reported to interact with the MTs as positive regulators of MT assembly (microtubule-associated proteins), either by promoting the polymerization of tubulin or by stabilizing MTs (1, 2). Only a few proteins are known to act as destabilizers, such as Op18͞stathmin, katanin, and some kinesin-like proteins (1, 2). We have reported recently that the M1 isoform of pyruvate kinase and Dictyostelium discoideum phosphofructokinase inhibit tubulin polymerization or promote disassembly of the MTs to thread-like oligomers in vitro (3-5).Recently, from bovine brain, we isolated and identified a protein that we denoted TPPP͞p25 (6), which corresponded to p25 (NCBI accession no. 2498194). This protein is a heat stable, cationic protein that induces polymerization of tubulin into unusual forms or bundling of paclitaxel-stabilized MT. The TPPP͞p25 protein was partially copurified with a tau kinase (7). The bovine TPPP͞p25-coding gene has been cloned (8), and recently the human homologue of TPPP͞p25 was cloned and mapped to the p15.3 region of chromosome 5. The identity between the bovine and human TPPP͞p25 proteins is 90% (9). This protein was also described as p24, a heat-resistant glycogen synthase kinase-3 inhibitor (10). It is important to note that TPPP͞p25 differs completely from the extensively characterized protein p25, which is a truncated form of p35 that deregulates cyclin-dependent kinase-5 activity by causing prolonged activation and m...

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confidence: 99%

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation

Tirián

Hlavanda

Oláh

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

116

136

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“…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]).…”

Section: Most Glycolytic Enzymes Bind Mtsmentioning

confidence: 99%

“…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]. VHL, a protein that contributes to HIF-1a degradation, is also a MT assembly regulator that stabilizes MTs without depleting the pool of tubulin dimers [Thoma et al, 2010].…”

Section: Metabolic Enzymes and Regulators Also Contribute To Regulatimentioning

confidence: 99%

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

et al. 2012

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Microtubules (MTs) or their subunits, tubulin dimers, interact with multiple components that contribute to intracellular metabolic pathways. MTs are required for insulin-dependent transport of glucose transporter 4 to the plasma membrane, they bind most glycolytic enzymes and are required for translation of the mRNA encoding hypoxia inducible factor-1a. Tubulin dimers bind the voltage-dependent anion channel of the mitochondrial outer membrane; this channel functions in metabolite transport in and out of mitochondria. We hypothesize that tubulin partitioning between dimer and polymer pools regulates multiple steps in metabolism, where metabolic output is greatest when both tubulin dimers and MT polymers are present and reduced by drug treatments that disrupt this normal balance. Experimental evidence from these drug-induced changes in tubulin dimer/polymer partitioning supports our model for several metabolic steps. Signal transduction pathways that stabilize or destabilize MTs can shift the normal ratio between unpolymerized and polymerized tubulin dimers, and one downstream consequence of this shift in tubulin partitioning could be a change in metabolic output. V C 2012 Wiley Periodicals, Inc Key Words: glycolysis, oxidative phosphorylation, glucose transport, voltage-dependent anion channel, tubulin IntroductionT he microtubule (MT) cytoskeleton has well-characterized roles in cell polarity, motility, mitosis, and vesicle traffic. The dynamic turnover of MTs is critical to most of these processes, allowing the cell to reorganize an array of MTs rapidly for specific functions. Here we posit an additional role for MTs in regulating metabolic processes and propose that metabolism is greatest when both MTs and their tubulin subunits are present in cells. Disruption of this normal balance, to either inhibit or promote MT polymerization, is expected to decrease metabolic output. Our hypothesis predicts that chemotherapies that stabilize or destabilize MTs will significantly impact metabolic pathways and reduce ATP levels. Importantly, even a small decrease in ATP production can slow cell proliferation. For example, a 19% decrease in ATP production was sufficient to induce senescence in mouse embryo fibroblasts [Kondoh et al., 2005], indicating that even a relatively small contribution of the MT cytoskeleton to metabolic output could have a significant impact on cell fate.The metabolic components and pathways we consider here include glucose transporters (GLUTs), glycolysis, the pentose phosphate pathway (PPP), and mitochondrial oxidative phosphorylation. Glucose enters cells through glucose transporters called GLUTs; the concentration of GLUTs at the plasma membrane can be regulated by insulin and requires trafficking of GLUT-containing vesicles along MTs to reach the plasma membrane. Within the cytoplasm, glucose is broken down to pyruvate during glycolysis, yielding ATP and NADH. Several intermediate products of glucose breakdown, including glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (GAP), can also ...

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Section: Microtubules (Mt)mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Kovács

Lőw

Pácz

et al. 2003

Journal of Biological Chemistry

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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...

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Phosphofructokinase from Dictyostelium discoideum Is a Potent Inhibitor of Tubulin Polymerization

Cited by 14 publications

References 51 publications

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation

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

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

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