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)

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubulebinding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.
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PMID:Two kinesins from Arabidopsis, KatB and KatC, have a second microtubule-binding site in the tail domain. 1724 81

The cytoplasmic distribution of cellular structures is known to depend on the balance between plus- and minus-end-directed motor complexes. Among the plus-end-directed kinesins, kinesin-1 and -2 have been implicated in the outward movement of many organelles. To test for a role of kinesin-1 previous studies mostly relied on the overexpression of dominant-negative kinesin-1 constructs. The latter are often cytotoxic, modify the microtubule network and indirect effects related to altered microtubule dynamics should be excluded. In the present study we present a novel kinesin-1 construct, encompassing the first 330 amino acids of kinesin heavy chain fused to GFP (kin330-GFP) that does not alter microtubules upon its overexpression. Kin330-GFP functionally inhibits kinesin-1 because it induces the peri-nuclear accumulation of mitochondria and intermediate filaments. Using this construct and previously established siRNA-mediated knock-down of kinesin-2 function, we assess the role of both motors in the subcellular distribution of distinct steps of the vaccinia virus (VV) life cycle. We show that kinesin-1, but not kinesin-2, contributes to the specific cytoplasmic distribution of three of the four steps of VV morphogenesis tested. These results are discussed with respect to the possible regulation of kinesin-1 during VV infection.
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PMID:Kinesin-1 plays multiple roles during the vaccinia virus life cycle. 1739 62

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, M396-734 corresponding to amino acids Q396-N734 was fused with 6xHis-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of M396-734 is not activated by Ca2+, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 microM, much higher than that of other kinesins reported. The cosedimentation assay indicated that M396-734 could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.
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PMID:Cotton GhKCH2, a plant-specific kinesin, is low-affinitive and nucleotide-independent as binding to microtubule. 1792 6

Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dynein's proposed transport and tethering functions in the cell.
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PMID:Kinesin and dynein-dynactin at intersecting microtubules: motor density affects dynein function. 1822 30

Kinesin-1 is a molecular transporter that trafficks along microtubules. There is some evidence that kinesin-1 targets specific cellular sites, but it is unclear how this spatial regulation is achieved. To investigate this process, we used a combination of in vivo imaging of kinesin heavy-chain Kif5c (an isoform of kinesin-1) fused to GFP, in vitro analyses and mathematical modelling. GFP-Kif5c fluorescent puncta localised to a subset of microtubules in live cells. These puncta moved at speeds of up to 1 microm second(-1) and exchanged into cortically labelled clusters at microtubule ends. This behaviour depended on the presence of a functional motor domain, because a rigor-mutant GFP-Kif5c bound to microtubules but did not move along them. Further analysis indicated that the microtubule subset decorated by GFP-Kif5c was highly stable and primarily composed of detyrosinated tubulin. In vitro motility assays showed that the motor domain of Kif5c moved detyrosinated microtubules at significantly lower velocities than tyrosinated (unmodified) microtubules. Mathematical modelling predicted that a small increase in detyrosination would bias kinesin-1 occupancy towards detyrosinated microtubules. These data suggest that kinesin-1 preferentially binds to and trafficks on detyrosinated microtubules in vivo, providing a potential basis for the spatial targeting of kinesin-1-based cargo transport.
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PMID:Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells. 1833 49

Despite the high level of similarity in structural organisation of their motor domains and, consequently, in the mechanism of motility generation, kinesin-5 moves about 25-fold slower than conventional kinesin (kinesin-1). To elucidate the structural motifs contributing to velocity regulation, we expressed a set of Eg5- and KIF5A-based chimeric proteins with interchanged native neck linker and neck elements. Among them, the chimera consisting of the Eg5 catalytic core (residues 1-357) fused to the KIF5A linker and neck (residues 326-450) displayed increased velocity compared to the Eg5 control protein. This is the first evidence that the velocity of the slow-moving motor Eg5 can be elevated by insertion of neck linker and neck elements taken from a fast-moving motor. Whereas the complementary chimera composed of the KIF5A core (1-325) and the Eg5 linker and neck (358-513) was found to be immotile, insertion of the first half-KIF5A linker into this chimera restored motility. Our results indicate that the neck linker and the neck are involved not only in motility generation in general and in determination of movement direction, but also in velocity regulation.
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PMID:The role of Kinesin neck linker and neck in velocity regulation. 1864 Jan 25

Conventional kinesin (Kinesin-1) is a microtubule-based molecular motor that supports intracellular vesicle/organelle transport in various eukaryotic cells. To arrange kinesin motors similarly to myosin motors on thick filaments in muscles, the motor domain of rat conventional kinesin (amino acid residues 1-430) fused to the C-terminal 829 amino acid residues of catchin (KHC430Cat) was bacterially expressed and attached to catchin filaments that can attach to and arrange myosin molecules in a bipolar manner on their surface. Unlike the case of myosin where actin filaments move toward the center much faster than in the opposite direction along the catchin filaments, microtubules moved at the same speed in both directions. In addition, many microtubules moved across the filaments at the same speed with various angles between the axes of the microtubule and catchin filament. Kinesin/catchin chimera proteins with a shorter kinesin neck domain were also prepared. Those without the whole hinge 1 domain and the C-terminal part of the neck helix moved microtubules toward the center of the catchin filaments significantly, but only slightly, faster than in the opposite direction, although the movements in both directions were slower than those of the KHC430Cat construct. The results suggest that kinesin has substantial mechanical flexibility within the motor domain, possibly within the neck linker, enabling its interaction with microtubules having any orientation.
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PMID:Direction and speed of microtubule movements driven by kinesin motors arranged on catchin thick filaments. 1864 44

XB-S is a protein with an amino-terminal-truncated form of tenascin-X (TNXB). However, the precise roles of XB-S in vivo are unknown. In this study, to determine the role of XB-S in vivo, we screened XB-S-binding proteins. FLAG-tagged XB-S was transiently introduced into 293T cells. Then its associated proteins were purified by immunoprecipitation using an anti-FLAG antibody and its components were identified by mass spectrometric analyses. Mitotic motor kinesin Eg5 was identified in the immunoprecipitates. XB-S and Eg5 proteins were co-localized in the cytoplasm in interphase and mitosis, but XB-S did not localize on mitotic spindle microtubules, on which Eg5 prominently localized in mitosis. As for Eg5 binding to XB-S, glutathione S-transferase-fused XB-S expressed in vitro directly bound to full-length Eg5 translated in reticulocyte lysate, and the XB-S-binding region was located in the motor domain of Eg5. Furthermore, during cell cycle progression XB-S showed a similar expression profile to that of Eg5. These results suggest possible involvement of XB-S in the function of Eg5.
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PMID:Truncated form of tenascin-X, XB-S, interacts with mitotic motor kinesin Eg5. 1867 83

Matrix metalloproteinases (MMPs) are endopeptidases that cleave matrix, soluble and membrane-bound proteins and are regulated by their endogenous inhibitors the tissue inhibitors of MMPs (TIMPs). Nothing is known about MMP/TIMP trafficking and secretion in neuronal cells. We focussed our attention on the gelatinases MMP-2 and MMP-9, and their inhibitor TIMP-1. MMPs and TIMP-1 fused to GFP were expressed in N2a neuroblastoma and primary neuronal cells to study trafficking and secretion using real time video-microscopy, imaging, electron microscopy and biochemical approaches. We show that MMPs and TIMP-1 are secreted in 160-200 nm vesicles in a Golgi-dependent pathway. These vesicles distribute along microtubules and microfilaments, co-localise differentially with the molecular motors kinesin and myosin Va and undergo both anterograde and retrograde trafficking. MMP-9 retrograde transport involves the dynein/dynactin molecular motor. In hippocampal neurons, MMP-2 and MMP-9 vesicles are preferentially distributed in the somato-dendritic compartment and are found in dendritic spines. Non-transfected hippocampal neurons also demonstrate vesicular secretion of MMP-2 in both its pro- and active forms and gelatinolytic activity localised within dendritic spines. Our results show differential trafficking of MMP and TIMP-1-containing vesicles in neuronal cells and suggest that these vesicles could play a role in neuronal and synaptic plasticity.
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PMID:Vesicular trafficking and secretion of matrix metalloproteinases-2, -9 and tissue inhibitor of metalloproteinases-1 in neuronal cells. 1881 73

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca(2+)-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca(2+), while both dimeric and tetrameric configurations in the presence of Ca(2+). Further, Ca(2+)-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca(2+).
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PMID:Kinesin-Calmodulin fusion protein as a molecular shuttle. 1988 Mar 75


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