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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cold-stable and cold-labile microtubules were prepared by two cycles of assembly and disassembly and two periods of exposure to cold. The cold-labile preparations were shown to contain a higher proportion of a high molecular mass microtubule-associated protein (MAP 2) than cold-stable preparations. The cold-stable preparations showed a much higher alkaline phosphatase activity. Stimulation of microtubule assembly by zinc led to increases in both cold stability and alkaline phosphatase activity.
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PMID:Differences in polypeptide composition and enzyme activity between cold-stable and cold-labile microtubules and study of microtubule alkaline phosphatase activity. 671 30

We have isolated a monoclonal antibody (150) that recognizes a phosphorylation epitope on the microtubule-associated protein (MAP) 1B. Immunoblot analysis of the developing rat central nervous system shows that monoclonal antibody 150 is directed against a protein of approximately 325 kDa (MAP 1B) that copolymerizes with microtubules through successive cycles of temperature-dependent assembly and disassembly. Furthermore, immunoprecipitated MAP 1B contains the epitope recognized by monoclonal antibody 150. Removal of phosphate from blotted proteins using alkaline phosphatase abolishes the binding of monoclonal antibody 150 to MAP 1B, indicating that the epitope is phosphorylated. In the developing rat nervous system, immunohistochemistry with monoclonal antibody 150 shows that the phosphorylation epitope on MAP 1B is transiently expressed in growing axons but not in dendrites. For instance, in the neonatal rat cerebellum, the parallel fibres of granule cells are stained only during elongation and not after synaptogenesis. The monoclonal antibody 150 epitope is also transiently expressed in radial glial fibres and in certain cell nuclei. All immunostaining of sections with monoclonal antibody 150 was completely abolished by alkaline phosphatase treatment. These observations and previous ones made by us in cell culture (Mansfield et al., J. Neurocytol., 20, 654-666, 1991) suggest that the phosphorylation epitope on MAP 1B recognized by monoclonal antibody 150, which has not been previously detected in vivo, may be important in axonogenesis.
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PMID:A phosphorylation epitope on MAP 1B that is transiently expressed in growing axons in the developing rat nervous system. 750 6

Microtubule-associated protein 1b, previously also referred to as microtubule-associated protein 5 or microtubule-associated protein 1x, is a major component of the juvenile cytoskeleton, and is essential during the early differentiation of neurons. It is required for axonal growth and its function is influenced by phosphorylation. The distribution of microtubule-associated protein 1b in kitten cerebellum and cortex during postnatal development was studied with two monoclonal antibodies. Hybridoma clone AA6 detected a non-phosphorylated site, while clone 125 detected a site phosphorylated by casein-kinase II. On blots, both monoclonal antibodies stained the same two proteins of similar molecular weights, also referred to as microtubule-associated protein 5a and 5b. Antibody 125 detected a phosphorylated epitope on both microtubule-associated protein 1b forms; dephosphorylation by alkaline phosphatase abolished the immunological detection. During development of cat cortex and cerebellum, AA6 stained the perikarya and dendrites of neurons during their early differentiation, and especially labelled newly generated axons. The staining decreased during development, and axonal staining was reduced in adult tissue. In contrast to previous reports which demonstrated that antibodies against phosphorylated microtubule-associated protein 1b label exclusively axons, antibody 125 also localized microtubule-associated protein 1b in cell bodies and dendrites, even in adulthood. Some nuclear staining was observed, indicating that a phosphorylated form of microtubule-associated protein 1b may participate in nuclear function. These results demonstrate that microtubule-associated protein 1b is subject to CK2-type phosphorylation throughout neuronal maturation and suggest that phosphorylation of microtubule-associated protein 1b may participate in juvenile and mature-type microtubule functions throughout development.
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PMID:Differential phosphorylation of MAP1b during postnatal development of the cat brain. 776

We previously showed that neurofilaments interact with microtubules (MTs) via their high molecular weight subunits (NF-H) after alkaline phosphatase treatment. Here we studied the effects of phosphorylation of NF-H on this interaction. tau protein kinase II, Ser/Thr protein kinase, phosphorylated NF-H in the tail domain, decreased its electrophoretic mobility to a native level, and also restored its property to be less interactive with MTs. Phosphorylation by cAMP-dependent protein kinase caused no shift of electrophoretic mobility or dissociation from MTs. We conclude that the tail domain of NF-H directly interacts with the MT surface, and the interaction is regulated via phosphorylation of the tail domain of NF-H by Ser/Thr protein kinase like tau protein kinase II. To characterize the binding domain of NF-H on MTs, subtilisin digestion of MTs and competition analysis with the MT binding fragment of tau protein were performed. The dissociation constant of NF-H to subtilisin MTs was higher than that to intact MTs. The maximum binding of NF-H was reduced when tau fragments existed. These results revealed that the COOH-terminal region of tubulin is involved in the binding to NF-H, and the NF-H and microtubule-associated protein binding domains are closely apposed on the surface of MTs.
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PMID:Interaction of the tail domain of high molecular weight subunits of neurofilaments with the COOH-terminal region of tubulin and its regulation by tau protein kinase II. 822 79

The microtubule-associated protein MAP2 is essential for development of early neuronal morphology and maintenance of adult neuronal morphology. Several splice variants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as a neuronal marker. In this study we compared five monoclonal antibodies (MAbs) against MAP2. They show differences in the immunocytochemical distribution of MAP2 isoforms during development of the visual cortex and cerebellum of the cat. Local and temporal differences were seen with MAb AP18, an antibody directed against a phosphorylation-dependent epitope near the N-terminal end. In large pyramidal dendrites in visual cortex, the AP18 epitope remained in parts immunoreactive after treatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognized the central region of the MAP2b molecule, which is not present in MAP2c and 2d, and reacted with phosphorylation-independent epitopes. During the first postnatal week the immunostaining in cerebellum differed between antibodies in that some cellular elements in external and internal granular layers and Purkinje cells were stained to various degrees, whereas at later stages staining patterns were similar. At early stages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex and cerebellum. With progressing maturation, immunoreactivity became restricted to distal parts of apical dendrites of pyramidal cells and was absent from perikarya and finer proximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adult animals. This study demonstrates that the immunocytochemical detection of MAP2 depends on modifications such as phosphorylation and conformational changes of the molecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation and specific conformations in the molecule may be essential for modulating function and molecular stability of MAP2, and monoclonal antibodies against such sites may provide tools for studying the functional role of modifications.
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PMID:Changes of MAP2 phosphorylation during brain development. 853 43

Hyperphosphorylated tau proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that tau phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m1 muscarinic acetylcholine receptor. Stimulation of m1 receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased tau phosphorylation, as indicated by specific tau monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on tau phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of tau microtubule-associated protein.
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PMID:Activation of m1 muscarinic acetylcholine receptor regulates tau phosphorylation in transfected PC12 cells. 859 66

Microtubules are important for the growth and maintenance of stable neuronal processes and their organisation is controlled partly by microtubule-associated proteins (MAPs). MAP 1B is the first MAP to be expressed in neurons and plays an important role in neurite outgrowth. MAP 1B is phosphorylated at multiple sites and it is believed that the function of the protein is regulated by its phosphorylation state. We have shown that the monoclonal antibody (mAb) RT97, which recognises phosphorylated epitopes on neurofilament proteins, fetal tau, and on Alzheimer's paired helical filament-tau, also recognises a developmentally regulated phosphorylation epitope on MAP 1B. In the rat cerebellum, Western blot analysis shows that mAb RT97 recognises the upper band of the MAP 1B doublet and that the amount of this epitope peaks very early postnatally and decreases with increasing age so that it is absent in the adult, despite the continued expression of MAP 1B in the adult. We confirmed that mAb RT97 binds to MAP 1B by showing that it recognises MAP 1B immunoprecipitated from postnatal rat cerebellum using polyclonal antibodies to recombinant MAP 1B proteins. We established that the RT97 epitope on MAP 1B is phosphorylated by showing that antibody binding was abolished by alkaline phosphatase treatment of immunoblots. Epitope mapping experiments suggest that the mAb RT97 site on MAP 1B is near the N-terminus of the molecule. Despite our immunoblotting data, immunostaining of sections of postnatal rat cerebellum with mAb RT97 shows a staining pattern typical of neurofilaments with no apparent staining of MAP 1B. For instance, basket cell axons and axons in the granule cell layer and white matter stained, whereas parallel fibres did not. These results suggest that the MAP 1B epitope is masked or lost under the immunocytochemical conditions in which the cerebellar sections are prepared. The upper band of the MAP 1B doublet is believed to be predominantly phosphorylated by proline-directed protein kinases (PDPKs). PDPKs are also good candidates for phosphorylating neurofilament proteins and tau and therefore we postulate that the sites recognised by RT97 on these neuronal cytoskeletal proteins may be phosphorylated by similar kinases. Important goals are to determine the precise location of the RT97 epitope on MAP 1B and the kinase responsible.
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PMID:The neurofilament antibody RT97 recognises a developmentally regulated phosphorylation epitope on microtubule-associated protein 1B. 930 99

The neurodegenerative process in Alzheimer's disease (AD) has been suggested to occur as a consequence of microtubule disruption and subsequent loss of intracellular transport. Structural microtubule-associated proteins (MAPs) have been investigated for their role in the etiology of AD, but dynein, a force-producing MAP which mediates intracellular transport, has not been examined. In this report, dynein (MAP1C) immunoreactivity in AD brain tissue homogenates was observed increased 3.7-fold compared with control brain homogenate preparations. Similarly, NGF-differentiated PC12 cells cultured in the presence of soluble extracts prepared from AD brain tissue homogenates, exhibited an approximate 15-fold increase in dynein immunoreactivity compared to that of control brain tissue extracts. In contrast, AD clarified extracts had little effect upon "kinesin-like" protein immunoreactivity increased (approximately 2-fold); whereas, tau immunoreactivity was observed to be moderately increased (5-fold) over that of control brain extract treated PC12 cells. Chemical dephosphorylation and alkaline phosphatase treatment of AD extract-treated PC12 cell lysate prior to Western blotting resulted in complete loss of immunoreactivity, suggesting the dynein being monitored is a phosphorylated isoform. Furthermore, treatment of clarified brain tissue extracts with trypsin and (NH4)2SO4 suggests the endogenous elements giving rise to increased PC12 cell dynein intermediate chain immunoreactivity to be proteinaceous in nature. The observed increase in dynein intermediate-chain dynein immunoreactivity following exposure of neuronal cells to endogenous elements of AD brain may be reflective of dynein-microtubular array differences. Such an approach may be useful in assessing the effect of endogenous biomolecules on retrograde axonal transport in neuronal culture models.
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PMID:Effect of Alzheimer's brain extracts on dynein immunoreactivity in PC12 cells. 940 50

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.
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PMID:Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5. 1509 91

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.
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PMID:In vitro neuronal differentiation of cultured human embryonic germ cells. 1562 48


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