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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)-dependent protein kinase activity, and microtubule-associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2', 3', or 5' position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3',5'-monophosphorothioate (cAMPS). The (Sp)-cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)-cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP-stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP-dependent phosphorylation on functional properties in microtubules in general.
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PMID:In vitro phosphorylation of microtubule-associated protein 2: differential effects of cyclic AMP analogues. 299 22

Based on a theory that a norepinephrine-stimulated cascade of events resulting in an increase of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) modulates the state of plasticity for the receptive field property of visual cortical neurons, we have followed the ontogenetic changes in cAMP-stimulated phosphorylation of proteins in whole homogenates obtained from developing visual cortices of cats. In vitro phosphorylation was assayed with and without cAMP and the cAMP-dependent protein kinase, and the phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were counted for 32P incorporated from [gamma-32P]ATP. It was found that the regulatory subunits of the cAMP-dependent protein kinase are present and fully active by birth, whereas the synapsin content increases at a rate concomitant with synaptogenesis. These ontogenetic developments are not influenced by dark rearing (DR) from birth, a procedure which postpones the onset of the critical period (CP) for plasticity. By contrast, the cAMP-stimulatable phosphorylation of microtubule-associated protein 2 (MAP 2), which under normal rearing conditions increases from birth to the second month, is strongly modulated by the presence of light in the environment. After DR for various periods, kittens were subsequently exposed to light so as to trigger the onset of the CP that had been postponed. A few hours of light were sufficient to cause a large increase in the in vitro phosphorylation of MAP 2. This effect is not observed in the auditory cortex or the lateral geniculate nucleus of the same animals, or in the visual cortex of normally reared cats which were then dark reared in adulthood. But this effect was seen in the visual cortices of cats following 5 months of DR from birth, animals which by chronological age have passed the CP, presumably because the onset of the CP was extended by the DR procedure. The cAMP-dependent phosphorylation of MAP 2 (and its dephosphorylation) may be an important factor for determining the state of plasticity in the CP through its affecting the dendritic cytoskeletal organization involving tubulin and actin.
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PMID:Ontogenetic changes in the cyclic adenosine 3',5'-monophosphate-stimulatable phosphorylation of cat visual cortex proteins, particularly of microtubule-associated protein 2 (MAP 2): effects of normal and dark rearing and of the exposure to light. 299 45

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.
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PMID:Purified protein kinase C phosphorylates microtubule-associated protein 2. 302 25

Calmodulin-dependent protein kinase II (CaM kinase II) is associated with microtubule preparations and phosphorylates several endogenous proteins including microtubule-associated protein 2, tubulin, and an 80,000-dalton protein doublet (pp80). We now report that pp80 is identical to synapsin I by all criteria studied including molecular weight, isoelectric point, phosphopeptide mapping of cAMP- and calmodulin-dependent phosphorylated protein, comigration with authentic synapsin I, and sensitivity to digestion with collagenase. Synapsin I and CaM kinase II were found in association with both microtubule preparations and preparations enriched in neurofilaments. Antibodies to synapsin I specifically labeled neurofilaments prepared in vitro. Immunocytochemical studies on rat brain tissue demonstrated synapsin I immunoreactivity specifically associated with the neuronal cytoskeleton as well as synaptic vesicles. The observed synapsin I staining on cytoskeletal elements was considerably diminished or abolished by the inclusion of Triton X-100 in the staining solutions. These results indicate that synapsin I is associated with the cytoskeleton and may be an important link between cytoskeletal elements as well as between the cytoskeleton and membrane.
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PMID:Association of synapsin I with neuronal cytoskeleton. Identification in cytoskeletal preparations in vitro and immunocytochemical localization in brain of synapsin I. 308 74

Mammalian microtubule-associated protein 2 (MAP2) exists in high-molecular-weight (Mr approximately 280,000) and low-molecular-weight (Mr approximately 70,000) forms, with the latter protein being more abundant in embryonic brain homogenates than in preparations from mature brain (Riederer and Matus, 1985). In the current study, we have shown that avian MAP2 also exists as both high- (Mr approximately 260,000) and low-molecular-weight (Mr approximately 65,000) forms whose relative abundance changes during brain maturation, indicating a conserved function for these proteins during vertebrate neuronal morphogenesis. Using indirect immunohistochemistry, we have determined the cellular distribution of the high- and low-molecular-weight forms of MAP2 in the developing avian cerebellum. In the embryonic cerebellum, low-molecular-weight MAP2 is found in the external granular layer and in epithelial cells. High-molecular-weight MAP2 is found only in neurons that have commenced dendrogenesis, i.e., Purkinje cells and neurons within the internal granular layer. Thus, low-molecular-weight MAP2 is not only more abundant in embryonic nervous tissue than in the adult, but it also appears in glia and in differentiating neurons before the high-molecular-weight form. We have also shown that in the mature cerebellum high-molecular-weight MAP2 cannot be detected with monoclonal antibodies or polyclonal antisera in Purkinje cell dendrites. Polyclonal antisera against the regulatory subunit of the cAMP-dependent protein kinase, which is associated with MAP2 in the Purkinje cell dendrites of the rat, also fail to stain Purkinje cell dendrites in the mature quail cerebellum. This suggests that high-molecular-weight MAP2 may be necessary for the establishment of dendrites but is not necessary for the maintenance of dendritic form.
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PMID:The sequential appearance of low- and high-molecular-weight forms of MAP2 in the developing cerebellum. 319 90

Treatment of quiescent human embryonic lung fibroblastic cells (TIG-3) with 10 nM epidermal growth factor (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at 15 min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several growth factors, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human fibroblasts.
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PMID:Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts. 325 98

Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with 32Pi. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The 32P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extent phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. We conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.
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PMID:Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. 328 75

The cellular and subcellular distribution of the regulatory subunit RII of cAMP-dependent protein kinase was studied by light and electron microscopy immunocytochemistry in tissue sections from rat brain and in primary cultures of brain cells. RII immunoreactivity was present in most neurons, although at variable concentration. In addition, RII was also detectable in other cell types including glia, neuroepithelial cells, and cells of mesenchymal origin. In the cell cytoplasm, RII immunoreactivity was concentrated at certain sites. An accumulation of RII immunoreactivity was found in all RII-positive cells at the Golgi area, precisely at a region directly adjacent to one of the two major faces of the Golgi complex. RII was also highly concentrated in some microtubule-rich cell processes such as cilia and neuronal dendrites, but was below detectability in most axons. In neurons, its concentration in dendrites is consistent with the previously demonstrated high affinity interaction between RII and the dendritic microtubule-associated protein 2. In addition, RII was accumulated at basal bodies of cilia and at centrosomes, i.e., sites known to act as microtubule organizers. RII-labeled centrosomes, however, were visible only in cells where the Golgi complex had a pericentrosomal organization, and not in cells where the Golgi complex was perinuclear such as in neurons and glia in situ. We hypothesize that centrosomal RII is bound to the pericentriolar microtubule-organizing material and that this material remains associated with the trans region of the Golgi complex when the latter is no longer associated with the centrosome. Our results suggest a key but not obligatory role of cAMP in the Golgi-centrosomal area, the headquarters of cell polarity, mobility and intracellular traffic, and in the function of a subpopulation of microtubules.
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PMID:Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex. 352 3

Isolated microtubule-associated protein 2 (MAP2), tau factor, and tubulin were phosphorylated by a purified Ca2+, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and tau factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, tau factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, tau factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca2+, calmodulin system via phosphorylation of MAP2, tau factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.
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PMID:Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation. 391 51

The association of regulatory subunits (RII) of Type II cAMP-dependent protein kinase from bovine cerebral cortex (RII-B) and bovine cardiac and skeletal muscle (RII-H) with specific binding proteins in bovine brain cytosol and purified brain microtubules was demonstrated using a solid phase binding assay. RII-binding proteins present in bovine cerebral cortex were immobilized on nitrocellulose filters after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the filters with 32P-labeled regulatory subunits showed that both RII-B and RII-H interact with the 75,000-dalton calmodulin-binding protein (P75) and microtubule-associated protein 2 (MAP-2). However, significant differences in binding affinities and capacities were observed. RII-B displayed a higher affinity for P75 compared to RII-H while RII-H preferentially bound to MAP-2. Quantitation of radioactive RII bound to MAP-2 showed that MAP-2 bound 4-6 times more RII-H than RII-B. The differential binding affinities and capacities of RII-H and RII-B for MAP-2 were not affected by autophosphorylation since both phospho and dephospho forms of RII displayed the same binding characteristics. Competitive binding studies suggest that RII-H and RII-B bind to the same sites on MAP-2. The biochemical basis for the differential binding of RII-B and RII-H to the same sites of MAP-2 is unknown. However, other high affinity RII-binding proteins present in cerebral cortex (i.e. P75) might affect the affinity of RII-B for MAP-2. 32P-RI did not bind to P75 nor MAP-2 under the conditions used.
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PMID:Differential binding of the regulatory subunits (RII) of cAMP-dependent protein kinase II from bovine brain and muscle to RII-binding proteins. 394 17


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