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)

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.
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PMID:Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. 192 62

Two intermediary kinases in a protein serine/threonine kinase cascade that is triggered in the response of Swiss 3T3 cells to epidermal growth factor (EGF) have been identified. Several separable EGF-stimulated serine/threonine kinase activities were characterized in the preceding paper (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E.G. (1990) J. Biol. Chem. 265, 11487-11494). These were preincubated in various combinations in the presence of MgATP with chromatographic fractions from unstimulated cell extracts. Activation of the rate of phosphorylation of a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, was observed on preincubation of the breakthrough fraction from unstimulated cell extracts with either of two distinct EGF-stimulated kinase activities, each of which phosphorylated myelin basic protein. Kinetic analysis and fractionation by sizing gel chromatography demonstrated that two myelin basic protein kinase activities (of approximately 30 and approximately 50 kDa) represented the activating components in the mixtures whereas the unstimulated cell extract breakthrough gave rise in each case to the activated Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala peptide kinase activity of approximately 110 kDa. Inasmuch as the in vitro activation reactions required magnesium plus ATP and were reversed by protein phosphatase treatment, an activation mechanism involving phosphoryl transfer is suggested.
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PMID:Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro. 214 54

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.
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PMID:An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A. 215 67

Phosphotyrosyl-protein phosphatase activity of human erythrocyte cytosol can be resolved into two fractions by DEAE-cellulose chromatography followed by P-cellulose chromatography. Both 32P-Tyr-phosphatases are able to dephosphorylate 32P-Tyr of poly (Glu-Tyr) 4:1 but not angiotensin II and synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Gly, previously phosphorylated on tyrosine residues by rat spleen tyrosine-protein kinase. Both 32P-Tyr-phosphatase activities distinctly differ from either 32P-Ser-casein phosphatase activity or "acid" and "alkaline" p-nitrophenylphosphatase activities with regard to catalytic and physico-chemical properties such as substrate specificity, chromatographic behaviour, response to various effectors.
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PMID:Partial purification and characterization of phosphotyrosyl-protein phosphatase(s) from human erythrocyte cytosol. 242 49

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.
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PMID:Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C. 255 Apr 47

The catalytic subunit of the Ca2+/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) incorporating approximately 1 mol of phosphoryl group/mol of catalytic subunit, in agreement with a value previously reported (Hashimoto et al., 1988). Cyanogen bromide cleavage of radiolabeled CN followed by peptide fractionation using reverse-phase high-performance liquid chromatography yielded a single labeled peptide that contained a phosphoserine residue. Microsequencing of the peptide allowed both the determination of the cleavage cycle that released [32P]phosphoserine and the identity of amino acids adjacent to it. Comparison of this sequence with the sequences of methionyl peptides deduced from the cDNA structure of CN (Kincaid et al., 1988) allowed the phosphorylated serine to be uniquely identified. Interestingly, the phosphoserine exists in the sequence Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the putative CaM-binding site. The phosphorylated serine residue was resistant to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could be powerfully stimulated by effectors of CN phosphatase activity. The mechanism of dephosphorylation may be intramolecular since the initial rate was the same at phosphoCN concentrations of 2.5-250 nM.
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PMID:Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain. 255 15

Protein phosphatase T from rat liver, so termed due to its activity toward [32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565-8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the alpha-subunit of phosphorylase kinase. The Mr of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2Ao of protein phosphatase 2A.
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PMID:Identification of pseudo 'phosphothreonyl-specific' protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A. 282 78

Prior phosphorylation of its substrate has been shown to be important for substrate recognition by the protein kinase glycogen synthase kinase-3 (GSK-3). Phosphorylation of glycogen synthase by GSK-3 is known to be enhanced by the previous action of casein kinase II and the sequence -SXXXS(P)- was proposed as the minimal recognition determinant for GSK-3. The glycogen binding subunit of type 1 phosphoprotein phosphatase has been shown to be phosphorylated by cyclic AMP-dependent protein kinase at serine-13 in the sequence KPGFS(5)PQPS(9)RRGS(13)ESSEEVYV (F.B. Caudwell, A. Hiraga, and P. Cohen (1986) FEBS Lett. 194, 85-89). Inspection of the sequence revealed potential GSK-3 sites at residues 5 and 9. Using a synthetic peptide with the above sequence, we found that phosphorylation of serine-13 by cyclic AMP-dependent protein kinase permitted the recognition of serine-9 and serine-5 by GSK-3. The work provides another example of a substrate for GSK-3 and demonstrates that the action of GSK-3 is linked to the presence of phosphate in the substrate and not the action of any particular protein kinase. In the course of the analyses, a novel feature of trypsin cleavage of phosphopeptides was noted. In the sequence -SRRGS(P)- trypsin acted uniquely after the first arginine whereas in the sequence -S(P)RRGS(P)- it cleaved randomly at either arginine residue. The fact that GSK-3 could phosphorylate a peptide derived from a phosphatase subunit also raises the possibility that GSK-3 might be involved in controlling glycogen-associated type 1 phosphatase and, more generally, in mediating cyclic AMP control of protein phosphorylation in cells.
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PMID:Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1. 285 Jul 71

1. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates fatty acid synthesis from glucose in isolated adipocytes with a half-maximal effect at 0.72 microM. In seven batches of cells, the maximal effects of TPA and insulin were 8.5 +/- 1.1-fold and 27.1 +/- 2.1-fold respectively. Insulin also stimulated fatty acid synthesis from acetate 8.9 +/- 0.5-fold (three experiments), but TPA did not significantly increase fatty acid synthesis from this precursor. 2. In contrast to insulin, TPA treatment of isolated adipocytes did not produce an activation of acetyl-CoA carboxylase which was detectable in crude cell extracts. 3. The total phosphate content of acetyl-CoA carboxylase, isolated from adipocytes in the presence of protein phosphatase inhibitors, was estimated by 32P-labelling experiments to be 2.6 +/- 0.1 (5), 3.4 +/- 0.2 (5), and 3.8 +/- 0.2 (3) mol/mol subunit for enzyme from control, insulin- and TPA-treated cells respectively. Insulin and TPA stimulated phosphorylation within the same two tryptic peptides. 4. Purified acetyl-CoA carboxylase is phosphorylated in vitro by protein kinase C at serine residues which are recovered in three tryptic peptides, i.e. peptide T1, which appears to be identical with the peptide Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys phosphorylated by cyclic-AMP-dependent protein kinase, and peptides Ta and Tb, which have the sequences Ile-Asp-Ser(P)-Gln-Arg and Lys-Ile-Asp-Ser(P)-Gln-Arg respectively, and which appear to be derived from a single site by alternative cleavages. None of these correspond to the peptides whose 32P-labelling increase in response to insulin or TPA. Peptides Ta/Tb are not significantly phosphorylated in isolated adipocytes, even after insulin or TPA treatment. Peptide T1 is phosphorylated in isolated adipocytes, but this phosphorylation is not altered by insulin or TPA. 5. These results show that TPA mimics the effect of insulin on phosphorylation, but not activation, of acetyl-CoA carboxylase, i.e. that these two events can be dissociated. In addition, phorbol ester stimulates phosphorylation of acetyl-CoA carboxylase in isolated adipocytes, but this is not catalyzed directly by protein kinase C, and acetyl-CoA carboxylase does not appear to be a physiological substrate for this kinase.
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PMID:Insulin and phorbol ester stimulate phosphorylation of acetyl-CoA carboxylase at similar sites in isolated adipocytes. Lack of correspondence with sites phosphorylated on the purified enzyme by protein kinase C. 290 Jan 39

Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.
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PMID:Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. 301 43


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