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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)

Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.
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PMID:Kinesin and cytoplasmic dynein binding to brain microsomes. 140 Mar 64

Movement of membrane-bounded organelles to intracellular destinations requires properly oriented microtubules and force-generating enzymes, such as the microtubule-stimulated ATPase kinesin. Kinesin is a heterotetramer with two heavy chain (approximately 124-kDa) and two light chain (approximately 64-kDa) subunits. Kinesin heavy chains contain both ATP- and microtubule-binding domains and are capable of force generation in vitro. Functions of the light chains are undetermined, although evidence suggests they interact with membrane surfaces. We have used molecular genetic approaches to dissect the kinesin light chain structure. Three distinct kinesin light chain cDNAs were cloned and sequenced from rat brain, and they were found to result from alternative splicing of a single gene. Polypeptides encoded by these cDNAs are identical except for their carboxyl ends. Synthesis of multiple light chains, differing from one another in primary structure, could provide a means of generating multiple, functionally specialized forms of the kinesin holoenzyme.
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PMID:Molecular genetics of kinesin light chains: generation of isoforms by alternative splicing. 194 31

Multiple transcripts coding for kinesin light chain isoforms are present in the tissues of the squid Loligo pealii. Isoform diversity arises through alternative RNA splicing in the amino and carboxyl termini of the putative proteins. Comparison to rat and Drosophila proteins demonstrates a remarkable conservation of structural domains and regulatory motifs. We have identified a PEST domain that may be the site of degradative uncoupling of kinesin functions. Selective transcript distribution occurs in disparate tissues, suggesting an adaptation toward specialized functions. Expression is highest in the nervous system and some evidence for neural-specific transcripts is provided. In neurons, this may relate to the differential targeting of specific membrane-bound organelles such as synaptic vesicles.
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PMID:Kinesin light chains: identification and characterization of a family of proteins from the optic lobe of the squid Loligo pealii. 827 23

We have deduced the amino acid sequences of four sea urchin (Strongylocentrotus purpuratus; SP) kinesin light chain (KLC) isoforms (SPKLC 1-4) and compared them to rat brain light chain sequences. Examination of the SPKLC open reading frames (SPKLC1, 649; SPKLC2, 677; SPKLC3, 686; and SPKLC4, 451 amino acid residues) reveals that the first 500 or so residues of the KLCs are highly conserved but the C-terminal ends of rat and sea urchin light chains are divergent; SPKLCs 1, 2 and 3 share a highly basic, 86 residue C-terminal segment that is missing from the shorter rat light chains and SPKLC4. The insertion of 28 and 37 residue segments at residue 563 of SPKLCs 2 and 3, respectively, gives rise to sequence heterogeneity at the C-terminal ends of the sea urchin KLCs. C-terminal sequence differences between light chains may provide inter- and intraspecies differences in the functional properties of the presumptive cargo attachment elements of kinesin.
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PMID:Sequences of sea urchin kinesin light chain isoforms. 849 62

A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum. Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats. Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis. Attempts to identify a canonical kinesin heavy-chain gene or protein were unsuccessful, suggesting that a kinesin heavy chain may be absent or unnecessary for kinesin light-chain function in this eubacterium. Our findings establish that certain basal elements of eukaryotic cellular transport appear to be resident in eubacteria. We discuss the possibility that the eukaryotic kinesin light chain was acquired by lateral gene transfer.
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PMID:Kinesin light chain in a eubacterium. 921 72

The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.
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PMID:Immunochemical analysis of kinesin light chain function. 924 47

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n-ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH-reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.
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PMID:n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay. 925 2

Kinesin, a plus-end-directed microtubule motor protein, functions in concert with accessory factors that have been shown to regulate enzyme activity and may also provide cargo specificity. This report identifies teh 79-kDa kinesin-associated phosphoprotein as a phosphoisoform of kinesin light chain. Increased phosphorylation of this light chain isoform is sufficient to account for the increase in kinesin-mediated microtubule-gliding activity. Additionally, it was found that the degree of phosphorylation of this isoform is regulated by a 100-kDa kinase and 150-kDa type 1 phosphatase. Both the kinesin light chain kinase and phosphatase co-purify with the kinesin heavy chain, suggesting that kinesin exists in a large complex capable of self-regulation.
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PMID:Phosphotransferases associated with the regulation of kinesin motor activity. 931 51

We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995) J. Cell Biol. 131, 1275-1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an approximately 2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of approximately 120 kDa and a light chain of approximately 64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.
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PMID:Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. Selective binding of kinesin to detyrosinated tubulin and vimentin. 954 18

Native kinesin consists of two light chains and two heavy chains in a 1:1 stoichiometric ratio. To date, only one gene for kinesin light chain has been characterized, while a second gene was identified in a genomic sequencing study but not analyzed biochemically. Here we describe new genes encoding kinesin light chains in mouse. One of these light chains is neuronally enriched, while another shows ubiquitous expression. The presence of multiple kinesin light chain genes in mice is especially interesting, since there are two kinesin heavy chain genes in humans (Niclas, J., Navone, F., Hom-Booher, N., and Vale, R. D. (1994) Neuron 12, 1059-1072). To assess the selectivity of kinesin light chain interaction with the heavy chains, we performed immunoprecipitation experiments. The data suggested that the light chains form homodimers with no specificity in their interaction with the two heavy chains. Immunofluorescence and biochemical subfractionation suggested differences in the subcellular localization of the two kinesin light chain gene products. Although both kinesin light chains are distributed throughout the central and peripheral nervous systems, there is enrichment of one in sciatic nerve axons, while the other shows elevated levels in olfactory bulb glomeruli. These results indicate that the mammalian nervous system contains multiple kinesin light chain gene products with potentially distinct functions.
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PMID:Two kinesin light chain genes in mice. Identification and characterization of the encoded proteins. 962 22


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