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)

Full-length Drosophila kinesin heavy chain from position 1 to 975 was expressed in Escherichia coil (DKH975) and is a dimer. The sedimentation coefficient of DKH975 shifts from 5.4 S at 1 M NaCl to approximately 6.9 S at <0.2 M NaCl. This transition of DKH975 between extended and compact conformations is essentially identical to that for the heavy chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D., and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic property of the heavy chains and requires neither light chains nor eukaryotic post-translational modification. DKH960 undergoes a similar transition, indicating that the extreme COOH-terminal region is not required. More extensive deletions from the COOH-terminal (DKH945 and DKH937) result in a shift in the midpoint for the transition to lower salt concentrations. DKH927 and shorter constructs remaining extended even in the absence of added salt. Thus the COOH-terminal approximately 50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH2-terminal head/neck segments interact in a salt-dependent manner that is consistent with the compact conformer being produced by the interaction of domains from these regions of the heavy chain dimer. The microtubule-stimulated ATPase rate of DKH975 in the compact conformer is strongly inhibited compared with the rate of extended DKH894 (4 s-1 and 35 s-1, respectively, for kcat at saturating microtubules).
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PMID:Formation of the compact confomer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity. 1032 54

The successful execution of mitosis in mammalian cells requires the activities of numerous kinesin-like proteins. The Mitotic Kinesin-Like Protein-1 (MKLP-1) localizes to the spindle equator and is believed to participate in the separation of spindle poles during anaphase B. Injection of antibodies against MKLP-1 into dividing cells results in cell cycle arrest, suggesting that MKLP-1 is essential for mitosis. To further characterize MKLP-1, constructs encoding C-terminal domains of MKLP-1 were expressed as fusions to the green fluorescent protein and localized in HeLa cells. All constructs localized to the nucleus indicating the presence of at least one nuclear localization sequence in the C-terminus of the protein. C-terminal domains of MKLP-1 expressed in insect cells also localized to the nucleus as shown by subcellular fractionation. These proteins remained tightly associated with the nucleus following both detergent and salt extraction, suggesting a tight interaction with a component of the nucleus.
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PMID:Nuclear localization of C-terminal domains of the kinesin-like protein MKLP-1. 1040 13

We have compared the interaction of ncd (non-claret disjunctional), a kinesin related protein, with microtubules and tubulin heterodimer. Ultracentrifugation experiments revealed that the ncd motor domain, residues 335-700 (ncd335), does not induce tubulin polymerization but stabilizes pre-formed microtubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 binding to tubulin or microtubules was estimated by following the change in fluorescence polarization of an exogenous dye attached to Cys670 of ncd335. Ncd335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06-0.08 sec(-1) and 1.8-2.0 sec(-1) for the ncd335-tubulin and ncd335-microtubules complexes, respectively. Only the polymerized complex is fully functional, suggesting the presence of additional contacts between adjacent protofilaments. Moreover, the data reveal that the oligomeric state of microtubules is a potent regulator for the activity of kinesin related proteins.
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PMID:Regulation of ncd by the oligomeric state of tubulin. 1062 29

Conventional kinesin is a highly processive molecular motor that takes several hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two heads of the kinesin dimer, but the specific factors that determine kinesin's run length (distance traveled per microtubule encounter) are not known. Here, we show that the neck coiled-coil, a structure adjacent to the motor domain, plays an important role in governing the run length. By adding positive charge to the neck coiled-coil, we have created ultra-processive kinesin mutants that have fourfold longer run lengths than the wild-type motor, but that have normal ATPase activity and motor velocity. Conversely, adding negative charge on the neck coiled-coil decreases the run length. The gain in processivity can be suppressed by either proteolytic cleavage of tubulin's negatively charged COOH terminus or by high salt concentrations. Therefore, modulation of processivity by the neck coiled-coil appears to involve an electrostatic tethering interaction with the COOH terminus of tubulin. The ability to readily increase kinesin processivity by mutation, taken together with the strong sequence conservation of the neck coiled-coil, suggests that evolutionary pressures may limit kinesin's run length to optimize its in vivo function.
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PMID:Engineering the processive run length of the kinesin motor. 1108 10

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.
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PMID:Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules. 1170 93

Kinesin binds to microtubules with half-site ADP release to form a tethered intermediate with one attached head without nucleotide and one tethered head that retains its bound ADP. For DKH405 containing amino acid residues 1-405 of Drosophila kinesin, release of the remaining ADP from the tethered head is slow (0.05 s(-1)), but release is accelerated by added ADP or ATP. The maximum rate of ADP-stimulated dissociation of tethered DKH405 from the microtubule is approximately 12 s(-1) as determined by turbidity. Parallel measurements of ADP-stimulated release of 2'(3')-O-(N-methylanthraniloyl)-ADP (mantADP) from the tethered intermediate by fluorescence indicate that the reaction is biphasic with a fast phase that occurs at a rate that is similar to dissociation. The rate of the slow phase is dependent on the concentrations of salt and microtubules and is equal in each case to the rate for bimolecular stimulation of ADP release by microtubules as measured independently. These results are consistent with a scheme in which the fast phase, with approximately one-third of the total amplitude change, is due to ADP-stimulated release of mantADP from the tethered intermediate at approximately 6 s(-1). This direct release of mantADP continues until terminated by dissociation of DKH405 from the microtubule at approximately 12 s(-1). The majority of the amplitude change thus occurs through bimolecular recombination of DKH405.mantADP with microtubules following initial dissociation. Analysis of a simple scheme indicates that hydrolysis of ATP at the attached head before the tethered head can release its ADP and become tightly bound may be the principal limitation to processivity.
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PMID:Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity. 1191 91

Previous studies have shown that West Nile (Sarafend) virus matured by budding at the plasma membrane, which differs from the usual intracellular maturation of other flaviviruses. The present study investigated the trafficking mechanism of the envelope (E) and capsid (C) proteins of West Nile (Sarafend) virus during the replication cycle. The use of time-based double-immunofluorescence labelling coupled with the Triton X-100 extraction procedure revealed that both the E and C proteins were transported from the perinuclear region towards the plasma membrane along the microtubules simultaneously. The strong association of these virus proteins with the microtubules was demonstrated further with Triton X-100 extraction procedure coupled with double immunogold-labelling. Extraction of infected cells with Triton X-100 in high salt also revealed that virus E proteins were associated with the microtubules via protein-protein interaction. The disruption of microtubules with vinblastine sulphate inhibited the trafficking of both the virus E and C proteins. Both virus structural proteins were observed to co-localise and retained within vinblastine sulphate-induced microtubulin paracrystals. Extracellular virus production was also reduced drastically by vinblastine sulphate at non-cytotoxic concentration. Subsequent studies revealed that the transportation of virus E protein was associated with the microtubules-based motor protein, kinesin.
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PMID:Trafficking mechanism of West Nile (Sarafend) virus structural proteins. 1192 Aug 27

The kinesin family member BimC has a highly positively charged domain of approximately 70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by approximately 7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.
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PMID:The kinesin family member BimC contains a second microtubule binding region attached to the N terminus of the motor domain. 1453 Feb 65

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

Microfilaments and microtubules (MT) play a vital role in cellular endocytic processes. The present study evaluates the role of these cytoskeletal elements in the apical internalization and postendocytic fate of riboflavin (RF) in placental trophoblasts (BeWo cells). Biochemical modification of the actin and microtubule network by (1) okadaic acid (OA), which disrupts MT-based vesicular trafficking; (2) cytochalasin D and latrunculin B, which promote actin depolymerization; and (3) 2,3-butanedione monoxime (BDM), which inhibits myosin-actin interaction, was confirmed by immunofluorescence microscopy using actin- and tubulin-specific antibodies. Furthermore, involvement of the molecular motors dynein and kinesin was assessed in the presence of (1) sodium orthovanadate, which inhibits dynein-ATPase activity and (2) adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt hydrate, a non-hydrolyzable ATP analog, which results in defective kinesin-driven processes. RF internalization consequent to cytoskeletal alterations was compared with that of a clathrin-dependent endocytic marker ([125I]-transferrin [TF]), a caveolae-mediated endocytic substrate ([3H]-folic acid [FA]), and a fluid-phase endocytic marker ([125I]-horse radish peroxidase [HRP]). Apical recycling and bidirectional transport of RF and TF was measured following cytoskeletal alterations. Results indicate that uptake of RF, TF, FA and HRP are markedly reduced (approximately 30-65%) in the presence OA and BDM, suggesting differential sensitivities to modification of kinesin-driven microtubules. However, actin depolymerization negatively affected HRP endocytosis alone, while RF, FA and TF internalization remained unchanged. Disturbances in protein phosphorylation cascades also influenced apical recycling while net ligand transport across monolayers remained unaffected. In conclusion, apical RF trafficking in placental cells is tightly regulated by microtubules and supported by accessory actin involvement.
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PMID:Cytoskeletal scaffolds regulate riboflavin endocytosis and recycling in placental trophoblasts. 1656 24


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