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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)

Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.
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PMID:Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging. 134 51

Ciliary or flagellar movement is the model of microtubule-dependent motility, the best studied at the molecular level. It is based on the relative sliding of outer doublets of microtubules that are linked at their proximal end to the basal structure and interconnected by associated proteins, among which dynein ATPase is at the origin of the movement. It is regulated from inside and outside media by various diffusible factors such as Ca2+, cyclic adenosine monophosphate (cAMP), polypeptides and so on (see other conferences presented during this meeting). Other motility processes are based on microtubules: vesicle and organelle transport through the cytoplasm (axonal flow in neurons, pigment granule movements in fish chromatophores, movements of particles along heliozoan axopods, etc.) could be mediated by microtubule motors such as kinesin or MAP 1C. Kinesin and MAP 1C, like dynein, are proteins that bind to microtubules and show an ATPase activity associated with force production. They differ from each other by their structure, and biochemical and pharmacological properties. The movements of chromosomes during mitosis and meiosis have long been studied, but are still poorly understood at the molecular level; this topic will be discussed in the light of recent data. Other constituents of the cytoskeleton are certainly involved in cellular motility: actin microfilaments and their motor myosin, intermediate filaments, non-actin filaments, all organized around the Microtubule Organizing Center (MTOC). As more information becomes available, it seems increasingly obvious that these various networks are closely interconnected and that each component probably modulates, resists, or favors properties of its partners, contributing to cellular and intracellular motility.
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PMID:From cilia and flagella to intracellular motility and back again: a review of a few aspects of microtubule-based motility. 246 57

We have analyzed the effects of various substrates and inhibitors on the rates of microtubule (MT) motility induced by sea urchin egg kinesin using real-time computer analysis and video-enhanced light microscopy. In the presence of magnesium, 10 mM concentrations of all the nucleotides tested supported MT translocation, with velocities in MgATP greater than MgGTP greater than MgTTP approximately equal to MgUTP greater than MgCTP greater than MgITP. The velocity of kinesin-driven MT motility is fairly uniform over approximately 3 pH units, from pH 6 to 9, with almost no motility outside this range. In the presence of ATP, no motility is observed in the absence of divalent cations; addition of Mg2+ but not addition of Ca2+ restores motility. MgATP-dependent MT motility is reversibly inhibited by Mg-free ATP, EDTA, or tripolyphosphate, suggesting that Mg-free ATP is an inactive substrate analogue. MgATP and MgGTP both obey saturable, Michaelis-Menten kinetics, with apparent Km values of approximately 60 microM and 2 mM, and Vmax values of approximately 0.6 and 0.4 microns/s, respectively. MgATP gamma S and MgADP are classic competitive inhibitors of kinesin-driven motility in MgATP, with Ki values of approximately 15 and 150 microM, respectively. Adenosine 5'-(beta, gamma-methylene)-triphosphate and N-ethylmaleimide only inhibit MT motility weakly, while adenyl-5'-yl imidodiphosphate and vanadate strongly inhibit MT motility, but not in a simple competitive manner. Moreover, in contrast to other inhibitors which cause a unimodal decrease in MT mean velocity, vanadate concentrations greater than approximately 10% that of MgATP cause some MTs to become immotile, resulting in a bimodal distribution of MT velocities.
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PMID:Quantitative analysis of sea urchin egg kinesin-driven microtubule motility. 252 43

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.
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PMID:Temperature-dependent reversible assembly of taxol-treated microtubules. 289 14

Recently, a protein called kinesin was described, which is capable of inducing movement of inert particles along microtubules. To purify this protein from bovine brain, we used the ability of kinesin to bind to taxol-stabilized microtubules in the presence of inorganic tripolyphosphate. The brain kinesin preparation contained one major polypeptide of 135 kDa and four minor polypeptides of 45-70 kDa. The minor polypeptides were eluted from a gel-permeation chromatography column at the same position as the major component. All the polypeptides of the preparation were capable of binding to the microtubules under identical conditions. The kinesin molecule is most probably a complex of these polypeptides. Brain kinesin had a very low ATPase activity (0.06-0.08 mumol X min-1 X mg-1 in 3 mM Mg2+ at pH 6.7). ATPase activity was strongly stimulated by microtubules (Vmax = 4.6 mumol per min per mg of kinesin). Microtubule-activated kinesin ATPase had a Km for ATP between 10 and 12 X 10(-6) M and a Kapp for microtubules (i.e., polymerized tubulin concentration required for a half-maximal activation) of 12-14 X 10(-6) M. Kinesin had a significant ATPase activity even without microtubules if 2 mM Ca2+ was substituted for Mg2+ (Vmax = 1.6 mumol X min-1 X mg-1; Km = 800 X 10(-6) M). Kinesin is therefore a mechanochemical ATPase that is activated by microtubules.
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PMID:Bovine brain kinesin is a microtubule-activated ATPase. 294 42

Sea urchin embryos in second division have been lysed into microtubule-stabilizing buffers to yield mitotic cytoskeletons (MCSs) that consist of two mitotic spindles surrounded by a cortical array of filaments. Microtubules have been completely extracted from MCSs by incubation at 0 degrees C with Ca2+-containing buffer. An antibody to the microtubule translocator kinesin stains the spindles in MCSs and in MCSs treated with 5 mM ATP and also stains spindle-remnants of the MCSs after the microtubules have been extracted. We conclude that kinesin binds to a nonmicrotubule component in the mitotic spindle. Based on these results, we present several models of kinesin function in the spindle.
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PMID:Kinesin is associated with a nonmicrotubule component of sea urchin mitotic spindles. 310 77

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.
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PMID:Purification and characterization of ensconsin, a novel microtubule stabilizing protein. 787 51

After injury to the cell membrane, rapid resealing of the membrane occurs with little loss of intracellular contents. This process has been studied by measurement of the rate of dye loss after membrane puncture in both the sea urchin embryo and 3T3 fibroblasts. Resealing of disrupted cell membranes requires external calcium that can be antagonized by magnesium. Block of multifunctional calcium/calmodulin kinase, which regulates exocytotic vesicle availability at synapses, and of kinesin, which is required for outward-directed transport of vesicles, inhibited membrane resealing. Resealing was also inhibited by botulinum neurotoxins B and A, suggesting that the two synaptosomal-associated proteins synaptobrevin and SNAP-25 also participate in resealing. This pattern of inhibition indicates that the calcium-dependent mechanisms for cell membrane resealing may involve vesicle delivery, docking, and fusion, similar to the exocytosis of neurotransmitters.
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PMID:Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. 790 84

Kinesin is an ubiquitous heterotetrameric microtubule-based motor which translocates membrane-bound organelles. Since organelle motility and motor protein function can be regulated by components of signaling pathways, the ability of purified bovine brain kinesin (kinesin) to be phosphorylated and to recognize calmodulin (CaM) was tested. Extensively purified "kinesin" was found to consist of several forms of both heavy (KHC) and light (KLC) chains. Phosphorylation of kinesin by a variety of protein kinases was examined; cAMP-dependent protein kinase (cAMP-PK) was the most active enzyme leading to the incorporation of up to 8 mol P/mol kinesin. Phosphorylation occurred predominantly on the KLCs and led to substantial acidic pI shifts. Peptide maps indicated that multiple phosphorylation sites exist on each KLC. Incubation of kinesin in vitro with protein kinase C (PKC) led to the phosphorylation of both KHCs and KLCs. In vivo phosphorylation of KHC and KLCs was demonstrated by immunoprecipitation of [32P]-labeled kinesin from cultured rat hippocampal pyramidal neurons; kinesin phosphorylation was stimulated by 8-chlorophenyl-thio-cAMP or 12-O-tetradecanoylphorbol-13-acetate. Native bovine brain kinesin was shown to bind 125I-CaM by nucleotide-dependent pelleting with stable microtubules. Specific calcium-dependent binding of 125I-CaM to KLCs but not KHC was found using a ligand blotting assay. cAMP-PK phosphorylated kinesin bound 125I-CaM less well than untreated protein in both ligand blotting and microtubule-pelleting paradigms. Calcium-dependent binding of CaM to kinesin inhibited the ATPase activity of native kinesin but not of cAMP-PK phosphorylated kinesin. These results suggest that the KLCs have a regulatory function and integrate information coming from diverse signaling pathways to modulate the activity and function of kinesin.
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PMID:Calmodulin binding to and cAMP-dependent phosphorylation of kinesin light chains modulate kinesin ATPase activity. 838 85

Previous work has indicated that cytoplasmic dynein localizes primarily to lysosomes in cultured fibroblasts, consistent with a function for dynein in retrograde movement. We now show that dynein can be redistributed from a lysosome-associated pool to a more diffuse cytoplasmic pool upon shifting fibroblasts to culture medium lacking serum for several hours. This effect on dynein localization is readily reversed upon addition of serum, with a substantial return to a control appearance of punctate staining within 10 minutes. The serum effect appears to be selective for dynein, in that the localization of kinesin and the overall morphology of intracellular organelles does not change. However, the distribution of kinesin-positive vesicles and lysosomes does appear to be altered during serum starvation, in that these organelles are located to greater extents in the peripheral regions of the cell. Dynein is also associated with the mitotic apparatus, but this localization does not change in response to serum starvation. Removal of calcium from the extracellular medium also results in the loss of punctate dynein staining, which can be recovered upon addition of calcium to calcium-free medium. The redistribution of dynein observed under these experimental conditions may reflect the activity of a regulatory process controlling the association of dynein with organelles, thereby providing one means of modulating intracellular transport.
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PMID:Regulation of the intracellular distribution of cytoplasmic dynein by serum factors and calcium. 840 87


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