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

---

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

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.
...
PMID:Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle. 775 99

Kinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface. The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule. The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/alpha5) that corresponds topologically to the major actin-binding domain of myosin. Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores.
...
PMID:Microtubule interaction site of the kinesin motor. 924 95

Three loop structures called the P-loop, switch I loop and switch II loop of myosin are major components of its ATPase site, and share structural and functional homology with the loop structures in other ATPases and GTPases such as kinesin and G-protein. Using the alanine scanning mutagenesis, structure-function relationship of the switch I and switch II loops in Dictyostelium myosin II was examined. Based on crystal structures of Dictyostelium myosin motor domain, functions of each residue in those loops are discussed.
...
PMID:Structure-mutation analysis of the ATPase site of Dictyostelium discoideum myosin II. 994 64

Transgenic mice that express a mutant Cu/Zn superoxide dismutase (SOD1) gene have been provided a valuable model for human amyotrophic lateral sclerosis (ALS). We studied a possible impairment of fast axonal transport in transgenic mice carrying a Gly93-->Ala (G93A) mutant SOD1 gene found in human familial ALS (FALS). Left sciatic nerve was ligated for 6 h in transgenic (Tg) and age-matched wild-type (WT) mice. Immunohistochemical analyses were performed for accumulations of kinesin and cytoplasmic dynein on both sides of the ligation site. Clinical function and histology in the spinal cords, sciatic nerves and gastrocnemius muscles were also assessed. The mice were examined at an early asymptomatic stage (aged 19 weeks) and a late stage (30 weeks) just before the development of the symptoms. WT mice showed an apparent increase in immunoreactivities for kinesin and cytoplasmic dynein at proximal and distal of the ligation, respectively. In contrast, the young Tg mice showed a selective decrease of kinesin accumulation in the proximal of the ligation. The mice were asymptomatic with a mild histological change only in muscles. The old Tg mice showed a marked reduction of the immunoreactivity for kinesin and cytoplasmic dynein on both sides of the ligation. They had a significant loss of spinal motor neurons, relatively small myelinated fiber densities of sciatic nerves, and severe muscular changes. These results provide direct evidence that the SOD1 mutation leads to impaired fast axonal transport, particularly in the anterograde direction at an early, asymptomatic stage preceding loss of spinal motor neurons and peripheral axons. This impairment may contribute to subsequent selective motor neuron death in the present model implicated for human FALS.
...
PMID:Selective impairment of fast anterograde axonal transport in the peripheral nerves of asymptomatic transgenic mice with a G93A mutant SOD1 gene. 1008 67

Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in in vitro assays should clarify the role of individual motor proteins in complex cellular processes.
...
PMID:Allele-specific activators and inhibitors for kinesin. 1043 Sep 3

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.
...
PMID:ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility. 1081 95

The Hedgehog (Hh) signaling molecule is required for the development of numerous tissues in Drosophila. Within the cell, Hh signal transduction utilizes a large protein complex consisting of the Fused (Fu), Costal2 (Cos2), and Cubitis interruptus (Ci) proteins, but the functional interactions between these proteins are still largely uncharacterized. Using a baculovirus system, we demonstrate that the serine/threonine kinase Fu phosphorylates the kinesin-like protein Cos2 when coexpressed with Cos2. Coexpression of Cos2 and a kinase-inactive version of Fu eliminates the majority of Cos2 phosphorylation. We then show that the primary Fu-induced phosphorylation site of Cos2 is serine 572, whereas serine 931 is phosphorylated to a lesser extent. Mutation of serine 572 to alanine eliminates most, but not all, specific phosphopeptides of Cos2 when coexpressed with Fu. We also demonstrate that the phosphorylation pattern of Cos2 produced by baculovirus coexpression with kinase-dead Fu is almost identical to the phosphorylation pattern of Cos2 isolated from unstimulated S2 cells. Finally, the phosphorylation pattern of Cos2 produced by baculovirus coinfection with wild-type Fu is almost identical to that of Cos2 isolated from S2 cells stimulated by Hh, indicating that phosphorylation of serines 572 and 931 is a genuine Hh signaling event. This study clarifies the unique functions of Fu and Cos2 in Hh signal transduction and identifies only the second known phosphorylation site of a kinesin-like molecule.
...
PMID:Hedgehog-stimulated phosphorylation of the kinesin-related protein Costal2 is mediated by the serine/threonine kinase fused. 1193 82

In an effort to understand how specific structural features within the kinesin neck, a region of the heavy chain located between the catalytic core and stalk domains, may contribute to motor processivity (an ability to remain attached to the microtubule filament), we have prepared several synthetic peptides corresponding to the neck region of human conventional kinesin and determined their secondary structure content and stability by CD spectroscopy. Our results show that the coiled-coil dimerization domain within the human kinesin neck region corresponds to residues 337 to 369 in solution, and thus is in excellent agreement with the recent X-ray crystallographic structures of rat brain kinesin. Further, we show that the first and last heptads of this region are absolutely critical for creating the high stability and association of the dimeric structure. Interestingly, addition of the 7 N-terminal neck-linker residues (330-336) to the coiled-coil domain significantly increased its stability (Delta GdnHCl midpoint of 1 M or an increase of approximately 1.5 kcal/mol), indicating that a strong structural link exists between the neck-linker and coiled-coil region. Subsequent high-resolution structural analysis of the residues located at the junction of the neck-linker and coiled-coil revealed the presence of the two helix capping motifs, the capping box (a reciprocal interaction of Thr 336 with Gln 339) and the hydrophobic staple (a hydrophobic packing interaction of Leu 335 with Trp 340). Substitution of Leu 335 and Thr 336 (the capping residues) with Gly completely eliminated the increased stability of the coiled-coil region observed in the presence of the neck-linker residues. Correspondingly, substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure. Subsequent analysis of the X-ray structure and substitution analysis of Lys 341 revealed that Trp 340 makes an important interchain hydrophobic interaction with Lys 341 of the opposite chain. Taken together these results reveal that a set of strong intra- and inter-chain interactions made up of the helix "capping box," "hydrophobic staple," and the newly identified "Leu-Trp-Lys sandwich" motifs stabilize the kinesin neck coiled-coil structure, thus preventing it from fraying and unfolding.
...
PMID:Helix capping interactions stabilize the N-terminus of the kinesin neck coiled-coil. 1206 48

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.
...
PMID:Motor domain mutation traps kinesin as a microtubule rigor complex. 1261 54

A series of modifications of the junction of the neck linker and neck coil of dimeric Drosophila kinesin were constructed to determine the influence of head orientation and spacing on the ATPase kinetics. Ala(345) is the first residue in the coiled-coil of the neck, and its replacement with glycine or proline produces no significant change in the k(cat) or K(0.5(MT)) values for activation of their ATPase by microtubules (MTs) or in their k(bi(ratio)) value for the average number of ATP molecules hydrolyzed during a processive encounter with a MT. Addition or deletion of a single amino acid at the junction produces only modest changes with less than a 2-fold reduction in kinetic processivity. Insertion of a spacer of 6 or 12 additional amino acids at the neck linker junction increases the K(0.5(MT)) value by 3-4-fold with a corresponding decrease in kinetic processivity. The sliding velocities of all the mutant constructs under multimotor conditions are within 30% of the wild-type value. All the constructs with single residue changes exhibit half-site ADP release on binding to MTs. The constructs with long insertion, however, rapidly release both ADP molecules per dimer on binding to a MT, indicating that the steric constraints that prevent release of ADP from the tethered head of wild-type kinesin have been relieved by the long insertions. The constructs with long inserts have decreased kinetic processivity and dissociate from the MT during ATP hydrolysis 3-fold faster than wild-type.
...
PMID:Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups. 1455 32


1 2 3 Next >>

---