Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.
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PMID:Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. 1250 85

Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu164 was identified as an amino acid critical for kinesin function [Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514]. Glu164 is located at the beta-strand 5a/loop 8b junction of the catalytic core and projects toward the microtubule binding face in close proximity to key residues on beta-tubulin helix alpha12. Substitution of Glu(164) with alanine (E164A) results in a dimeric kinesin with a dramatic reduction in the microtubule-activated steady-state ATPase (5 s(-1) per site versus 22 s(-1) per site for wild-type). Our analysis shows that E164A binds ATP and microtubules with a higher affinity than wild-type kinesin. The rapid quench and stopped-flow results provide evidence that ATP hydrolysis is significantly faster and the precise coordination between the motor domains is disrupted. The data reveal an E164A intermediate that is stalled on the microtubule and cannot bind and hydrolyze ATP at the second head.
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PMID:Motor domain mutation traps kinesin as a microtubule rigor complex. 1261 54

In this study we extend our analysis of the effect of Cytochalasin J (CJ) on mitotic and interphase cells by the use of immunocytochemical techniques to localize antigens to anti-beta-tubulin, anti-dynein heavy chain (HC), anti-dynein intermediate chain (IC), and anti-kinesin antibodies following CJ treatment. Anti-dynein IC and HC staining of CJ treated cells showed a significant reduction in anti-dynein staining in the nuclear region of interphase cells. Monolayer cultures of PtK(1)cells treated with 10 microg/ml CJ for 10 min showed a significant reduction in pixel luminosity of fluorescence staining using anti-dynein IC and HC antibodies (P<0.05). Cytochalasin J treatment reorganized anti-dynein staining from a cytoplasmic punctate staining with greatest intensity in the perinuclear region, to a more uniform staining throughout the cytoplasm.
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PMID:Redistribution of motor proteins by Cytochalasin J treatment. 1286 58

Several human neurodegenerative diseases are associated with abnormal accumulations of aggregated tau proteins and glial degeneration in astrocytes, but the mechanism whereby tau proteins cause astrocytic degeneration is unclear. Here, we analyzed the biological consequences of overexpressing the longest human tau isoform in primary cultures of rat astrocytes using adenoviral-mediated gene transfer. Significantly, we found specific decreases in stable detyrosinated [glutamate (Glu)] microtubules (MTs) with concomitant increases in tubulin biosynthesis and the accumulation of acetylated, tyrosinated, alpha- and beta-tubulin. The consequences of this selective reduction in stable Glu-MTs included contemporaneous decreases in kinesin levels, collapse of the intermediate filament network, progressive disruption of kinesin-dependent trafficking of organelles, fragmentation of the Golgi apparatus that culminated in atrophy, and non-apoptotic death of astrocytes. These results suggest that reduced stable Glu-MTs is a primary consequence of tau accumulation that initiates mechanisms underlying astrocyte dysfunction and death in human neurodegenerative glial tauopathies.
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PMID:Reduction of detyrosinated microtubules and Golgi fragmentation are linked to tau-induced degeneration in astrocytes. 1462 51

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure. Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.
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PMID:Complex formation with kinesin motor domains affects the structure of microtubules. 1465 46

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.
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PMID:Microtubule-kinesin interface mutants reveal a site critical for communication. 1500 14

Cells of cultured line XL2 (Xenopus laevis) were synchronized by a combine effect of serum deprivation, aphidicolin, nocodazole and ALLN treatments. Four fractions were prepared, with maximum percentage of cells being in G1, S and G2 phases of cell cycle, and in mitosis, respectively. Comparative levels of six different proteins (beta-tubulin, DNA topoisomerase IIa, Xenopus Aurora A kinase pEg2, kinesin-like motor protein X1Eg5, and two members of condensis family proteins pEg7 (XCAP D2) and XCAP E were detected by quantitative Western blot analysis of these fractions. We used a new method of mathematic processing of data that commonly provides a possibility to calculate a comparative quantity of proteins in hypothetically "clean" fraction composed of cells being in the same phase of the cell cycle. This method makes it possible to use even partly synchronized cell cultures for analysis of changes in protein quantity, provided a precede determination of cell population composition is made.
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PMID:[A comparative level of expression of some proteins in XL2 cell synchronized on different phases of cell cycle]. 1521 70

We have studied the structure of microtubules decorated with kinesin motor domains in different nucleotide states by 3D electron microscopy. Having docked the atomic coordinates of both dimeric ADP.kinesin and tubulin heterodimer into a map of kinesin dimers bound to microtubules in the presence of ADP, we try to predict which regions of the proteins interact in the weakly binding state. When either the presence of 5'-adenylyimidodiphosphate (AMP-PNP) or an absence of nucleotides puts motor domains into a strongly-bound state, the 3D maps show changes in the motor domains which modify their interaction with beta-tubulin. The maps also show differences in beta-tubulin conformation compared with undecorated microtubules or those decorated with weakly-bound motors. Strongly-bound ncd appears to produce an identical change.
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PMID:3D electron microscopy of the interaction of kinesin with tubulin. 1521 83

The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.
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PMID:Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin. 1712 95

A stable MIN6 beta-cell clone overexpressing glucokinase as an enhanced cyan fluorescent protein (ECFP) fusion construct was generated for analysis of glucokinase regulation in these glucose-responsive insulin-secreting cells. A higher glucokinase enzyme activity accompanied by an improved glucose-induced insulin secretion indicated the integration of ECFP-glucokinase into the functional pool of glucokinase protein in MIN6-ECFP-glucokinase cells. Fluorescence recovery after photobleaching experiments of MIN6-ECFP-glucokinase cells and photoactivation of a transiently transfected photoswitchable cyan fluorescent protein (PS-CFP)-glucokinase construct in MIN6 cells indicate a higher motility of the diffusible glucokinase fraction at high glucose concentrations. In agreement with previous studies, we observed significant binding of ECFP-glucokinase to insulin secretory granules. Using fluorescence lifetime imaging, we obtained evidence for an association between glucokinase and alpha-tubulin in MIN6-ECFP-glucokinase cells. Furthermore, immunohistochemistry and fluorescence resonance energy transfer analysis by acceptor photobleaching showed distinct association between endogenous glucokinase and alpha-tubulin as well as beta-tubulin in MIN6 cells. Interestingly, glucokinase was also colocalized with kinesin, a motor protein involved in insulin secretory granule movement. Therefore, we suggest a role of a bound glucokinase protein fraction in the regulation of insulin granule movement along tubulin filaments.
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PMID:Novel insights into the regulation of the bound and diffusible glucokinase in MIN6 beta-cells. 1728 61


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