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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel peptide substrate for adenosine 3',5'-cyclic monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), Leu-Arg-Arg-Trp-Ser-Leu-Gly, was synthesized. Phosphorylation of the peptide causes a 20% increase in the peptide fluorescence intensity at 358 nm. Values of Km and kcat for the phosphorylation reaction at pH 7.0 (25 degrees C), were determined to be 2.7 +/- 0.5 microM and 5.5 +/- 0.4 sec-1, respectively. The phosphorylated peptide was shown to be an effective substrate for phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) with a Km of 113 +/- 10 microM and a kcat of 2.4 +/- 0.2 sec-1 in the presence of 2.5 mM MnCl2. Changes in the peptide fluorescence intensity as a function of its phosphorylation state provide a highly sensitive assay of cyclic AMP-dependent protein kinase and phosphoprotein phosphatase activities.
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PMID:Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities. 627 44

The NH2-terminal blocking group of the Ca2+-binding B-subunit of calcineurin (protein phosphatase-2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl-Gly-Asn-Glu-Ala-, is very similar to that of the catalytic subunit of cyclic AMP-dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.
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PMID:Identification of the NH2-terminal blocking group of calcineurin B as myristic acid. 716 Apr 76

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.
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PMID:Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A. 753 74

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.
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PMID:Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A. 753 75

1. Acute homologous desensitization of mu-opioid receptor-induced currents was pharmacologically characterized in locus coeruleus (LC) neurones by use of intracellular and whole cell recording in superfused brain slices. 2. Following desensitization of opioid receptors by perfusion with a high concentration of [Met5] enkephalin (ME) for 5 min, there was a reduction in the maximum response and a rightward shift of the concentration-response curves for ME, [D-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO) and normorphine. 3. By simultaneously fitting the operational model to the paired pre- and post-desensitization concentration-response data for each agonist, estimates of the level of desensitization were obtained. The values obtained for the three agonists (between 88% and 96%) were similar and did not vary according to the efficacy of the agonist used. 4. Use of whole cell patch recording techniques caused a slow rundown in the amplitude of ME currents (approx. 40% reduction over 60 min) but did not greatly affect the expression of acute desensitization of opioid currents. 5. When included in the patch recording solution, the phosphatase inhibitors, microcystin (50 nM-4 microM) and okadaic acid (1 microM) had no effect on the induction of desensitization or the normal ability of opioid or alpha 2-adrenoceptors to produce currents. Microcystin decreased the rate of recovery of the ME (300 nM) currents following desensitization; however, okadaic acid had little effect on the rate of recovery from desensitization. 6. Strong calcium buffering with BAPTA (10-20 mM) had no effect on desensitization or the recovery from desensitization. 6. Strong calcium buffering with BAPTA (10-20 mM) had no effect on desensitization or the recovery from desensitization.7 These results suggest that acute homologous desensitization of micro-opioid receptors in LC neurones entails a rapid loss of responsiveness that involves a majority of the receptor population. The mechanism by which desensitization is reversed may involve a non-calcium-dependent protein phosphatase but the processess that cause desensitization remain unclear.
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PMID:Characterization of acute homologous desensitization of mu-opioid receptor-induced currents in locus coeruleus neurones. 758 22

A unique and highly conserved structural feature of approximately 90-kDa ribosomal S6 kinase (p90rsk or RSK) is the presence of two non-identical kinase domains. To explore the mechanism of RSK activation, a cloned human RSK cDNA (RSK3) was used to generate and characterize several site-directed RSK mutants; K91A (N-Lys, NH2-terminal ATP-binding mutant), K444A (C-Lys, COOH-terminal ATP-binding mutant), N/C-Lys (double ATP-binding mutant) T570A (C-Thr, mutant of the putative MAPK phosphorylation site in subdomain VIII of the C-domain), S218A (N-Ser, mutant of the corresponding NH2-terminal residue). Epitope-tagged RSKs were expressed in transfected COS cells followed by immunoprecipitation with or without prior in vivo epidermal growth factor stimulation. Kinase activity (S6 peptide) of N/C-Lys and N-Lys was ablated (and partially impaired with N-Ser). In contrast, both C-Lys and C-Thr retained high levels of kinase activity and were capable of responding to stimulation. C-Lys also retained partial kinase activity toward other substrates (c-Fos, S40 ribosomes, protein phosphatase 1 G-subunit, histones, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)) whereas N-Lys did not. The isolated NH2-and COOH-terminal domains were also expressed; the C-domain was inactive, whereas the N-domain retained partial activity. Relative to wild-type, both N-Lys and C-Lys (as well as N-Ser and C-Thr) underwent partial in vitro autophosphorylation that was further stimulated by EGF protein tyrosine phosphatase. We conclude that 1) the NH2-terminal RSK kinase domain mediates substrate phosphorylation; 2) both domains contribute to autophosphorylation; 3) the putative MAPK phosphorylation site is not required for growth factor-stimulated autophosphorylation or kinase activation.
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PMID:Divergent functional roles for p90rsk kinase domains. 764 38

A chimeric mutant was constructed in which a 4-amino acid region (GEFD, residues 274-277) of rabbit muscle protein phosphatase-1 was replaced with the sequence YRCG corresponding to residues 267-270 of rabbit protein phosphatase-2A. This was based on the findings of a gene mutation in okadaic acid-resistant cells which results in a Cys-->Gly conversion in protein phosphatase-2A. The YRCG mutant of protein phosphatase-1 was expressed and purified. The properties of the mutant enzyme were investigated in terms of its sensitivity toward several toxin inhibitors (okadaic acid, microcystin, nodularin, calyculin A, and cantharidic acid), as well as inhibitor-2. The mutant enzyme exhibited a gain of function in the form of a 10-fold increased sensitivity toward okadaic acid that suggests this region is involved in toxin binding. Significant changes in sensitivity to inhibitor-2 and several of the other toxins were also observed, indicating that these may have a common binding region.
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PMID:A mutant of protein phosphatase-1 that exhibits altered toxin sensitivity. 800 4

Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.
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PMID:The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role. 882 89

The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is a phosphatase that can dephosphorylate tyrosine-containing peptides, Shc, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PKB) and to inhibit cell spreading and motility. It is currently unclear, however, whether PTEN accomplishes these effects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppression by PTEN. By using a PTEN G129E (Gly(129)-->Glu) mutant that has lost its lipid phosphatase activity, while retaining protein phosphatase activity, we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PSD95, Dlg and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK293 cells (human embryonic kidney 293 cells) assayed in vitro. Surprisingly, expression of this mutant revealed differential requirements for the C-terminus in the different functional assays. Wild-type and C-terminally deleted PTEN appeared to be equally active in down-regulating PKB activity, but this mutant enzyme had no effect on platelet-derived growth factor (PDGF)-induced membrane ruffling and was only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to mutations that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.
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PMID:Analysis of the cellular functions of PTEN using catalytic domain and C-terminal mutations: differential effects of C-terminal deletion on signalling pathways downstream of phosphoinositide 3-kinase. 1069 13

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.
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PMID:Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells. 1087 31


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