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)

Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster.
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PMID:Molecular and genetic characterization of SLC1, a putative Saccharomyces cerevisiae homolog of the metazoan cytoplasmic dynein light chain 1. 862 45

Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.
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PMID:Interaction of mant-adenosine nucleotides and magnesium with kinesin. 954 60

Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor through the course of its ATPase cycle. In this study, we report on the production of a series of single tryptophan mutants of a monomeric human kinesin motor domain, which demonstrate nucleotide-dependent changes in microtubule affinity that are similar to wild type. We have used these mutations to measure intramolecular distances in both strong and weak binding states, using fluorescence resonance energy transfer. This work provides direct evidence that movement of the switch II loop and helix are essential to mediate communication between the catalytic and microtubule binding sites, evidence that is supported as well by molecular modeling. Kinetic studies of fluorescent nucleotide binding to these mutants are consistent with these distance changes, and demonstrate as well that binding of ADP produces two structural transitions, neither of which are identical to that produced by the binding of ATP. This study provides a basis for understanding current structural models of the kinesin mechanochemical cycle.
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PMID:Kinesin has three nucleotide-dependent conformations. Implications for strain-dependent release. 1085 22

Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility. Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release. The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes. This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation. The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence. The increase was caused by conformational changes on ATP binding. The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays. This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins.
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PMID:F1-ATPase changes its conformations upon phosphate release. 1188 Mar 67

Detecting protein-protein interactions other than those of antibody-antigen pairs still represents a demanding and tedious task. In the present work, a novel method as an alternative to current molecular biology-based detection procedures is established. It solely relies on the change of fluorescence decay times of the protein's intrinsic fluorophores tryptophan and tyrosine due to protein-protein interaction. Unlike previously utilized related methods, no labelling of the binding partners is required. This opens the possibility to detect proteins and their natural interactions without perturbation due to chemical alteration. The technique uses immobilization of one of the protein partners onto solid supports, which allows performance of protein binding studies in the microarray format. Fluorescence lifetime experiments of proteins in their different binding states have been applied to protease/protease-substrate pairs, as well as to the tubulin/kinesin system. Different binding behavior of proteins in solution towards protein partners immobilized on protein microarrays was detected with regard to binding specificity and protein amount. This label-free method for analyzing protein microarrays offers broad applicability ranging from principal investigations of protein interactions to applications in molecular biology and medicine.
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PMID:Readout of protein microarrays using intrinsic time resolved UV fluorescence for label-free detection. 1517 39

Kinesin-1 microtubule motors are common kinesin motors from protozoa, fungi and animals. They transport vesicular or particle cargo in a strictly regulated manner. The relatively well-studied tail inhibition mechanism is based on a conformational change that leads to an interaction of Kinesin-1's tail with the junction of neck and hinge regions. This folding causes a decrease in microtubule binding and motor activity. In fungal Kinesin-1 motors several lines of evidence suggest that a conserved tyrosine in the neck coiled-coil mediates this inhibition. In the active state, a region surrounding a conserved tryptophan in the hinge stabilises the neck coiled-coil, and prevents the tyrosine from inhibiting. Although animal and fungal Kinesin-1 motors are clearly homologous and function according to the same chemo-mechanical mechanism, they differ in their regulation. Unlike fungal Kinesin-1s, animal kinesins associate with light chains that are important for regulation and cargo interaction. Several proteins interacting with animal Kinesin-1 heavy or light chains are known, among them typical scaffolding proteins that seem to link Kinesin-1 to signalling pathways.
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PMID:Review: regulation mechanisms of Kinesin-1. 1645 53

The TRP (transient receptor potential) superfamily of cation channels is present in all eukaryotes, from yeast to mammals. Many TRP channels have been studied in the nematode Caenorhabditis elegans, revealing novel biological functions, regulatory modes, and mechanisms of localization. C. elegans TRPV channels function in olfaction, mechanosensation, osmosensation, and activity-dependent gene regulation. Their activity is regulated by G protein signaling and polyunsaturated fatty acids. C. elegans TRPPs related to human polycystic kidney disease genes are expressed in male-specific neurons. The KLP-6 kinesin directs TRPP channels to cilia, where they may interact with F0/F1 ATPases. A sperm-specific TRPC channel, TRP-3, is required for fertilization. Upon sperm activation, TRP-3 translocates from an intracellular compartment to the plasma membrane to allow store-operated Ca2+ entry. The TRPM channels GON-2 and GTL-2 regulate Mg2+ homeostasis and Mg2+ uptake by intestinal cells; GON-2 is also required for gonad development. The TRPML CUP-5 promotes normal lysosome biogenesis and prevents apoptosis. Dynamic, precise expression of TRP proteins generates a remarkable range of cellular functions.
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PMID:TRP channels in C. elegans. 1646 Feb 89

Polycystin-2 (PC2), encoded by PKD2, which is one of the genes whose mutations cause polycystic kidney disease, is abundantly produced in the apical domain of the syncytiotrophoblast (hST) of term human placenta. PC2, a TRP-type (TRPP2) non-selective cation channel, is present in primary cilia of renal epithelial cells, a microtubule-based ancillary structure with sensory function. The hST has abundant cytoskeletal structures, and actin filament dynamics regulate PC2 channel function in this epithelium. However, it is expected that the apical hST excludes microtubular structures. Here, we demonstrated by Western blot and immunocytochemical analyses that hST apical vesicles indeed contain microtubule structural components, including tubulin isoforms, acetylated alpha-tubulin, and the kinesin motor proteins KIF3A and KIF3B. PC2 and tubulin were substantially colocalized in hST vesicles. Treatment of hST vesicles with either the microtubular disrupter colchicine (15 microM) or the microtubular stabilizer paclitaxel (taxol, 15 microM) resulted in distinct patterns of microtubular re-organization and PC2 redistribution. We also observed that changes in microtubular dynamics regulate PC2 channel function. Addition of colchicine rapidly inhibited PC2 channel activity in lipid-bilayer reconstituted hST membranes. Addition of either tubulin and GTP, or taxol, however, stimulated PC2 channel activity in control hST membranes. Interestingly, we found that the kinesin motor protein KIF3A was capable of increasing PC2 channel activity in hST. We believe that the data are the first to provide a direct demonstration of a microtubular interaction with PC2 in the hST. This interaction thus plays an important regulatory role in the control of ion transport in the human placenta.
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PMID:Polycystin-2 cation channel function in the human syncytiotrophoblast is regulated by microtubular structures. 1720 94

Alcadeinalpha (Alcalpha) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alcalpha strongly associates with kinesin light chain (K(D) approximately 4-8x10(-9) M) through a novel tryptophan- and aspartic acid-containing sequence. Alcalpha can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alcalpha, and the kinesin-1 motor complex dissociates from Alcalpha-containing vesicles in a JIP1 concentration-dependent manner. Alcalpha-containing vesicles were transported with a velocity different from that of amyloid beta-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alcalpha- and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alcalpha with kinesin-1 blocked transport of APP-containing vesicles and increased beta-amyloid generation. Inappropriate interactions of Alc- and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease.
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PMID:The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport. 1733 54

SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.
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PMID:Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis. 1980 40


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