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

---

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

Two antipeptide antibodies (designated type 1 antibody and type 2A antibody) were raised against synthetic peptides, Cys-Thr-Pro-Pro-Arg-Asn-Ser-Ala-Lys-Ala-Lys-Lys and Cys-Val-Thr-Arg-Arg-Thr-Pro-Asp-Try-Phe-Leu, corresponding to the carboxyl termini of the catalytic subunits of protein phosphatase 1 and phosphatase 2A (Cys was added for specific coupling to carrier protein). These antipeptide antibodies were highly specific and were useful in discriminating between protein phosphatase 1 and phosphatase 2A in crude extracts or purified preparations. Type 2A antibody reacted with both native and denatured protein phosphatase 2A whereas under the same condition type 1 antibody reacted only with denatured protein phosphatase 1.
...
PMID:Antibodies directed against synthetic peptides distinguish between the catalytic subunits of protein phosphatases 1 and 2A. 131 44

In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.
...
PMID:Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase. 152 87

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.
...
PMID:Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver. 153 38

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.
...
PMID:Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit. 164 86

Insulin action leads to the rapid stimulation of a cytosolic Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) kinase (KIK) that has been recently purified to near homogeneity (Klarlund, J. K., Bradford, A. P., Milla, M. G., and Czech, M. P. (1990) J. Biol. Chem. 265, 227-234). To examine its activation mechanism, purified KIK was treated with purified protein phosphatases. The catalytic subunit of phosphatase 2A inhibited the activity of control KIK by about 50% and abolished the 5-fold elevation in KIK activity due to insulin action. The catalytic subunit of phosphatase 1 with equivalent activity based on dephosphorylation of 32P-labeled phosphorylase alpha had no effect on either control or insulin-stimulated KIK activity. The deactivation of insulin-stimulated KIK by phosphatase 2A was time- and concentration-dependent and was blocked by phosphatase inhibitors. The purified native complexes of phosphatase 2A, phosphatase 2A1, and phosphatase 2A2 similarly deactivated KIK. Analyis of control or insulin-stimulated KIK with two antiphosphotyrosine antibodies by immunoblotting and immunoprecipitation failed to detect the presence of phosphotyrosine in the kinase. These results indicate that KIK is activated by phosphorylation as part of a kinase cascade emanating from insulin receptor stimulation.
...
PMID:An insulin-stimulated kemptide kinase purified from rat liver is deactivated by phosphatase 2A. 184 13

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.
...
PMID:Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin. 200 42

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.
...
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.
...
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.
...
PMID:Partial purification and characterization of phosphotyrosyl-protein phosphatase(s) from human erythrocyte cytosol. 242 49

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.
...
PMID:Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain. 255 15


1 2 3 4 5 6 7 8 9 10 Next >>

---