Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.
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PMID:A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator. 132 14

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.
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PMID:Okadaic acid, a protein phosphatase inhibitor, inhibits nerve growth factor-directed neurite outgrowth in PC12 cells. 132 35

Cytokines such as interleukin-1, which are found in the brain after trauma, regulate expression of nerve growth factor (NGF) mRNA and protein in hippocampal cultures. We have investigated possible mechanisms by which Il-1 beta regulates NGF in hippocampal cells. The induction of NGF mRNA by Il-1 beta was blocked by a receptor antagonist indicating that this effect is receptor mediated. Il-1 beta elicited a dramatic induction of c-fos mRNA and a slight elevation of c-jun mRNA in a time dependent manner which may allow for a role in the induction of NGF mRNA expression. We examined whether specific second messenger pathways were involved in mediating the action of Il-1 beta in the hippocampus. Activation of cAMP with forskolin or treatment with 8-Br-cAMP had no effect on NGF mRNA levels. Moreover, exposure of hippocampal cultures to Il-1 beta evoked no change in cAMP levels, indicating that this second messenger system played little or no role in the regulation of NGF expression by Il-1 beta in these cells. Further, interleukin-1 elicited no change in membrane inositol phosphate turnover, nor did it affect intracellular calcium levels. Treatment of cell cultures with the phorbol ester PMA elicited an increase in NGF mRNA, suggesting that activation of protein kinase C (PKC) may mediate NGF mRNA expression. However, prolonged treatment of cultures with PMA to desensitize PKC did not eliminate the Il-1 beta induction of NGF mRNA. Il-1 beta, therefore, did not appear to activate NGF expression via cAMP, Ca2+, or a PKC isoform that is downregulated by prolonged PMA treatment. However, a phosphorylation event may be involved in the signal transduction mechanism, as treatment with okadaic acid to inhibit protein phosphatase 2a potentiated the induction of NGF mRNA by Il-1 beta. The results presented indicate that Il-1 beta acts via its receptor to induce a rise in NGF expression. Identification of the specific second messenger pathway has remained elusive; however, a phosphorylation event appears to be intermediary. Moreover, the induction of c-fos and c-jun may represent a final common path in activation of NGF gene expression by different signals such as Il-1 beta and PMA.
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PMID:Mechanisms of nerve growth factor mRNA regulation by interleukin-1 beta in hippocampal cultures: role of second messengers. 133 37

Nerve growth factor stimulates the uptake of radioactive calcium into PC12 cells. This stimulation is inhibited by low concentrations of dideoxyforskolin or staurosporine, and by high concentrations of nifedipine or cadmium. On the other hand, neither dideoxyforskolin nor staurosporine inhibited the stimulation of calcium uptake caused by BK-8644 or adenosine triphosphate (ATP). Nickel inhibited only the effect of ATP on calcium uptake, and actually stimulated the effects of either BK-8644 or nerve growth factor. Down-regulation of L-calcium channels by BK-8644 blocked the subsequent stimulation of calcium uptake by this agent, but not the stimulation by nerve growth factor. Conversely, pre-treatment of the cells with nerve growth factor inhibited the subsequent stimulation of calcium uptake by nerve growth factor, but not the stimulation by BK-8644. The effects of BK-8644 and nerve growth factor on calcium uptake were additive, as were the effects of nerve growth factor and ATP. Phosphatase 2A inhibited the effect of nerve growth factor on calcium uptake, but did not influence the action of BK-8644. On the other hand, calcineurin inhibited the effect of BK-8644 on calcium uptake, but potentiated the action of nerve growth factor. Calmidazolium or fluphenazine also inhibited the effect of nerve growth factor on calcium uptake, but okadaic acid stimulated it. A comparison of the effects of these inhibitors on the actions of various calcium channel agonists shows that the channels on which the action of nerve growth factor is exerted are different than either the L-type calcium channels or the ATP-activated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nerve growth factor-stimulated calcium uptake into PC12 cells: uniqueness of the channel and evidence for phosphorylation. 137 75

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter.
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PMID:Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells. 164 6

Calmodulin-dependent phosphoprotein phosphatase (CaMDP) activity has been found in each of three cultured cell lines: rat pheochromocytoma (PC12), glioma (C6), and pituitary adenoma (GH3) cells. These CaMDP activities bind to immobilized calmodulin in the presence of Ca2+ and are eluted by EGTA. Sucrose density centrifugation revealed that the phosphatase activities exhibited sedimentation coefficients of 4.37, 4.23, and 4.59 for proteins derived from C6, GH3, and PC12 cells, respectively. The Stokes radii measured for the PC12 and C6 activities were 41.8 and 40.0 A, respectively. The estimated molecular weights calculated for the enzymes from these data are 79,100 and 72,200. The phosphatase activities required the presence of divalent cations such as Ca2+ or Mn2+ for expression of activity, which was optimal only in the presence of calmodulin. The apparent Km for phosphorylated myelin basic protein substrate was 8 microM. Affinity-purified antibodies to the B subunit of bovine brain CaMDP were found by immunoblot (Western blot) to cross-react with a single protein among proteins extracted from PC12, C6, and GH3 cells that had been resolved by two-dimensional electrophoresis. In each case, the cross-reacting protein exhibited an Mr of 16,000 and an isoelectric point of 4.7, values virtually identical to those reported previously for the B subunit of bovine brain CaMDP (sometimes called calcineurin). This cross-reacting protein was found among cellular proteins eluted from immobilized calmodulin by EGTA. Immunocytochemical localization of the cross-reacting protein in undifferentiated PC12 cells or in cells differentiated in response to nerve growth factor revealed its presence diffusely throughout the cytoplasm. These experiments support the contention that each of these cell lines contains a calmodulin-regulated phosphatase homologous physically and kinetically, and immunologically related to bovine brain CaMDP.
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PMID:Calmodulin-dependent phosphatases of PC12, GH3, and C6 cells: physical, kinetic, and immunochemical properties. 329 45

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.
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PMID:Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells. 358 24

The immunosuppressant drug FK506 acts by binding to receptor proteins, FK506-binding proteins (FKBPs), which in turn can bind to and regulate a Ca(2+)-dependent phosphatase, calcineurin, and a Ca2+ release channel, the ryanodine receptor. Based on our findings in regeneration models that levels of FKBPs during neural regeneration parallel those of growth-associated protein GAP43, a calcineurin substrate that regulates neurite extension, we examined effects of FK506 in PC12 rat pheochromocytoma cells and in rat sensory ganglia. FK506 enhances neurite outgrowth in both systems by increasing sensitivity to nerve growth factor. Blockade of FK506 actions in sensory ganglia by rapamycin, an FK506 antagonist, establishes that these effects involve FKBPs. Rapamycin itself stimulates neurite outgrowth in PC12 cells. These drug effects are detected at subnanomolar concentrations, suggesting therapeutic application in diseases involving neural degeneration.
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PMID:Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia. 751 27

K-252a treatment produced a 30-50% increase in the uptake of radioactive calcium by PC12 cells within 3-4 minutes. The increase in uptake was partially blocked by inhibitors of voltage-operated calcium channels, such as nifedipine, but not by inhibitors of receptor-operated calcium channels, such as nickel or suramin. Introduction of phosphatase 2A into the cells completely blocked the effect of K-252a. Long-term treatment of the cells with either K-252a or with nerve growth factor blocked the subsequent actions of either K-252a or nerve growth factor on calcium uptake, but neither altered the subsequent action of the L-channel agonist Bay K 8644 on calcium uptake. Calcium uptake was not stimulated by K-252a in PC12nnr, cells that have little or no high-affinity nerve growth factor receptors; cells expressing increased levels of high-affinity nerve growth factor receptors showed a response to K-252a comparable to that seen in parent PC12. The data suggest that the increased uptake of radioactive calcium produced by K-252a is mediated by a mechanism very similar to that serving the increased calcium uptake produced by nerve growth factor.
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PMID:Characteristics of the K-252a-induced increase in calcium uptake in PC12 cells. 761 9

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 microM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation-sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 microM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 microM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 microM okadaic acid treatment; similar results were seen using the calcineurin-selective inhibitor cypermethrin. Treatment of human slices with 10 microM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau-directed kinase, 42-kDa mitogen-activated protein kinase (p42mapk), was activated by okadaic acid (> 100 nM) but not FK-520 (5 microM). Nerve growth factor (100 ng/ml) activated p42mapk, particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 microM) antagonized the effects of nerve growth factor on both p42mapk activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.
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PMID:Tau phosphorylation in brain slices: pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase. 772 35


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