Glycolytic enzyme–tubulin interactions: Role of tubulin carboxy terminals

Völker, Katharina; Knull, Harvey R.

doi:10.1002/jmr.300060405

Cited by 23 publications

(11 citation statements)

References 65 publications

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“…Both processes were suppressed under high salt conditions and were absent in subtilisin-treated MT samples. These data are in good agreement with earlier observations demonstrating the sensitivity of PK-tubulin interaction to salt concentration (15) and to proteolytic cleavage of the Cterminal segment of tubulin (16). The mechanism of formation of oligomeric aggregates needs further elucidation.…”

Section: Table I Effect Of Mt Assembly On the Distribution Of Pk In Bsupporting

confidence: 91%

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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Section: Table I Effect Of Mt Assembly On the Distribution Of Pk In Bsupporting

confidence: 91%

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

Kovács

Lőw

Pácz

et al. 2003

Journal of Biological Chemistry

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“…Numerous studies have demonstrated interactions between the cytoskeleton and glycolytic enzymes, especially GAPDH, aldolase and PK. [25][26][27][28][29][30][31][32][33][34][35][36][37][38] In this study, quantities of these glycolytic enzymes, as well as actin, tubulin and tropomyosin, were sufficient in U87 pseudopodia to be detected with Figure 4 DeCyder ratios Coomassie blue staining, thus establishing the potential for the energy-generating portion of the glycolytic pathway to energize cytoskeletal proteins for migration within U87 pseudopodia. Polymerization of actin and tubulin shares a similar use of energy from nucleotide triphosphate hydrolysis.…”

mentioning

confidence: 66%

Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies

Beckner

Chen

et al. 2005

Laboratory Investigation

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Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 lm pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from twodimensional (2D) gels with M R 's of 20-100 kDa and pI's of 3.0-10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) a chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion.

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“…MTs are known to decrease the overall activities of glyceraldehyde-3-phosphate dehydrogenase (11), aldolase (12,13) and PFK (14). The MT-bound dehydrogenase and PFK are inactive due to the preferential binding of the dissociated inactive forms of the enzymes to the MTs (11,14).…”

Section: Microtubules (Mt)mentioning

confidence: 99%