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Query: UNIPROT:P06889 (Mol)
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The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highest levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide beta-II (422-434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the beta-II tubulin fragment, but it showed interaction with the Affigel-conjugated beta-I (431-444) tubulin peptide. The different MAPs components were characterized by western blots using specific monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1994 Feb 23
PMID:Purification and characterization of the high molecule weight microtubule associated proteins from neonatal rat brain. 803 75

The role of insulin receptor tyrosine kinase activity in stimulation of intracellular enzymes linked to insulin action [phosphatidylinositol 3-kinase (PtdIns 3-kinase), microtubule-associated protein (MAP) kinase, and S6 kinases] was studied in Chinese hamster ovary cells which overexpress wild type human insulin receptors, receptors with reduced kinase activity due to substitution of Phe for Tyr1146 (single-Phe), Tyr1150,1151 (double-Phe), and Tyr1146,1150,1151 (triple-Phe), or kinase-inactive receptors with a substitution of Ala for Lys1018 in the ATP binding site (A1018). We have previously shown that receptor autophosphorylation and kinase activity of these mutants were reduced by approximately 50, 65, 85, and 100%, respectively. Glycogen and DNA synthesis parallel the level of receptor autophosphorylation and kinase activity; however, receptor serine and threonine phosphorylation was independent of receptor tyrosine kinase activity and receptor internalization was completely dependent on maximal receptor kinase activity. Overexpression of the wild type insulin receptor increased both maximal insulin receptor substrate-1-associated and total insulin-stimulated PtdIns 3-kinase activity, as well as S6 and MAP kinase activities 2.0- to 3.6-fold. In addition there was a leftward shift of the dose-response curves for PtdIns 3-kinase and S6 kinases by approximately 10-fold. Expression of the single- and double-Phe mutant receptors also enhanced maximal PtdIns 3-kinase activity, but had no effect on insulin sensitivity, whereas expression of either the triple-Phe or kinase-inactive receptors did not enhance insulin stimulation or increase insulin sensitivity as compared to the control cells. When comparing the mutant and wild type receptors, differences in insulin sensitivity were least for insulin-stimulated MAP kinase and greatest for S6 kinase; with the latter there was greater than a 1000-fold difference in insulin sensitivity when cells that overexpress wild type vs. kinase-inactive insulin receptors were compared. Thus, the level of insulin receptor tyrosine autophosphorylation and kinase activity regulate both maximal activation and insulin sensitivity of these intracellular kinases in the insulin action pathway which may lead to glycogen and/or DNA synthesis. The differential sensitivity of these enzymes to changes in receptor activation suggests that they may be differently coupled to the receptor kinase.
Mol Endocrinol 1994 May
PMID:The level of insulin receptor tyrosine kinase activity modulates the activities of phosphatidylinositol 3-kinase, microtubule-associated protein, and S6 kinases. 805 65

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.
Mol Cell Biol 1993 Dec
PMID:A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette. 824 79

Tau protein is a neuronal microtubule-associated protein that promotes the assembly and stability of microtubules. To evaluate the biological significance of tau isoform diversity, NIH-3T3 cells were stably transfected with cDNAs encoding each of the six isoforms present in human brain. Cells expressing different isoforms developed distinct morphologies. Cell lines expressing 3-repeat tau isoforms developed large flat cell bodies while cells expressing 4-repeat isoforms had small, round cell bodies. All transfected cell lines, except those expressing the shortest tau isoform, displayed very long thin neurite-like processes. Tau colocalized with microtubules in both the cell body and the long processes in all of the tau-transfected cells. Tau also displayed a diffuse amorphous staining pattern that was concentrated around the cell nucleus. Microtubule bundling was not enhanced in any of the transfected cells as compared to untransfected controls. The transfected cells showed increased resistance to colchicine treatment. Thus, different tau isoforms can confer unique cellular morphologies to 3T3 cells and can alter the susceptibility of these cells to a microtubule depolymerizing agent.
Brain Res Mol Brain Res 1993 Nov
PMID:Human tau isoforms confer distinct morphological and functional properties to stably transfected fibroblasts. 830 59

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.
Mol Biol Cell 1993 Mar
PMID:Dynamics of microtubules from erythrocyte marginal bands. 848 21

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.
Mol Biol Cell 1995 Oct
PMID:Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells. 857 94

DMAP190 is a microtubule-associated protein from Drosophila that is localized to the centrosome. In a previous study, we used affinity chromatography to identify proteins that interact with DMAP190, and identified a 60-kDa protein that we named DMAP60 (Kellogg and Alberts, 1992). Like DMAP190, DMAP60 interacts with microtubules and is localized to the centrosome, and the two proteins associate as part of a multiprotein complex. We now report the cloning and sequencing of the cDNA encoding DMAP60. The amino acid sequence of DMAP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for DMAP60 maps to chromosomal region 46A. In agreement with others working on Drosophila centrosomal proteins, we have changed the names for DMAP190 and DMAP60 to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase or early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. We find that bacterially expressed CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation.
Mol Biol Cell 1995 Dec
PMID:CP60: a microtubule-associated protein that is localized to the centrosome in a cell cycle-specific manner. 859 Jul 97

Tau, a microtubule-associated protein, is encoded by a single gene, the expression of which is neuron-specific and developmentally regulated. When PC12 cells are exposed to nerve growth factor (NGF), they differentiate to sympathetic-like neurons. This differentiation process is accompanied by an elevation of tau proteins and mRNA. Here, we describe, for the first time, the isolation and characterization of a tau promoter region. We show that the promoter of tau is G + C-rich, lacks a genuine TATA box and thus promotes multiple initiation sites of RNA transcription. Our results demonstrate that a region of approximately 335 base-pairs residing immediately upstream of tau exon -1 are able to direct positive control of neuron-specific activity of the luciferase reporter gene. The isolation of tau promoter will facilitate facilitate further studies of the regulation of tau expression during development and aging of neuronal cells.
J Mol Biol 1996 Mar 15
PMID:Identification of a tau promoter region mediating tissue-specific-regulated expression in PC12 cells. 860 31

Microtubules are integral components of the cytoskeleton of human cells and are composed of alpha- and beta-tubulin as well as a variable number of microtubule-associated proteins. In monocytes and macrophages, microtubules bind endotoxin and partly regulate endotoxin-induced inflammatory events such as cytokine production. Endotoxin causes a rapid alteration in monocyte microtubule stability. To characterize the effect of endotoxin on mononuclear phagocyte microtubule composition, Western blots and flow cytometry were performed on human monocytes and the monocyte/macrophage-like cell line THP-1. Compared to unstimulated monocytes, monocytes stimulated with endotoxin for 18 h had increased quantities of alpha-, beta-, and tyrosinated alpha-tubulin as well as microtubule-associated protein-2. PMA-differentiated THP-1 cells had increased levels of alpha-tubulin, beta-tubulin, microtubule-associated protein-5, microtubule-associated protein-2, and tau after endotoxin stimulation. These results indicate that endotoxin can alter mononuclear phagocyte microtubules by causing an increase in certain microtubule component proteins.
Am J Respir Cell Mol Biol 1997 Feb
PMID:Changes in mononuclear phagocyte microtubules after endotoxin stimulation. II. Changes in microtubule composition. 903 19

Breakdown or disruption of the cytoskeleton has been implicated in the neurodegenerative processes of a variety of diseases, including Alzheimer disease (AD) and stroke. Studies of such diseases in the human involve the use of postmortem brain tissue. Postmortem delay may vary considerably from a few hours to a few days, and within this period, a degree of cytoskeletal breakdown may occur. It is therefore crucial to examine alterations occurring in the cytoskeleton as a result of postmortem delay and subtract these from those caused by the disease. In this study, the distribution of tau, MAP2, and MAP5 immunohistochemistry was examined following postmortem intervals of 0-72 h in the rat cerebral cortex, corpus callosum, caudate nucleus, and hippocampus. Each microtubule-associated protein (MAP) underwent unique changes that were dependent both on postmortem interval and the brain region examined. Following long postmortem delays, some of the changes in these proteins were similar to those seen in rodent models of cerebral ischemia. These results demonstrate that MAPs are not stable during postmortem delay in the rat. Therefore, caution must be exercised when interpreting changes in MAPs in human postmortem tissue, especially in cases where ischemic injury may be involved. Examination of control tissue carefully matched for postmortem delay is therefore essential to allow meaningful interpretation of cytoskeletal abnormalities in human neurodegenerative disease.
Mol Chem Neuropathol 1997 Apr
PMID:The effect of postmortem delay on the distribution of microtubule-associated proteins tau, MAP2, and MAP5 in the rat. 916 90


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