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)

In the IL-2-dependent murine cytotoxic T cell line CTLL-2, IL-2 induced a rapid and transient decrease in Co(2+)-trypsin-treated activity of protein phosphatase PP1. The PP1 activity declined to a minimum level, being 70% of control value, in 20 min after the addition of IL-2 but recovered to the control level within 45 min. The decrease of PP1 activity was dependent on IL-2 concentration and occurred specifically in cytosolic fraction. Similar alteration was observed in IL-2 sensitive murine T-lymphoblasts. Neither activity of protein phosphatase PP2A nor PP2C showed alteration during the IL-2 stimulation. These results suggest that PP1 plays an important role in early events of the intracellular growth signaling from the IL-2 receptor.
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PMID:IL-2 induces transient and specific decrease in cytosolic protein phosphatase PP1 activity in murine T cell lines. 839 34

In this study, we examined the developmental expression and regulation by insulin and insulin-like growth factor-I (IGF-I) of protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) in cultured fetal chick neurons. Protein phosphatase activities were measured using 32P-labeled phosphorylase-a or 32P-labeled S6 kinase substrate peptide. In cell extracts from day 1-5 cultures, 40-45% of spontaneous protein phosphatase activity was due to PP-1. PP-2A accounted for the remaining 55-60% of enzyme activity. Spontaneous PP-1 activity increased by 100% in day 2 cultures and remained constant thereafter. PP-2A activity increased by 48% in day 2 cultures, with minimal increases in enzyme activity in later cultures. Under the assay conditions employed, at all times in culture a significant proportion (45-50%) of PP-1 was in an inactive form that could be reactivated by trypsin. PP-2A activity was not influenced by trypsin. Insulin stimulated neuronal PP-1 activity in day 4 and 5 cultures, but had no effect in earlier cultures. The activation of PP-1 by insulin was rapid, with a maximal effect (30-40% increase over basal levels) at 5 min with 10 ng/ml insulin. Insulin did not alter total (trypsin-released) PP-1 activity, the content of PP-1 catalytic subunit, or PP-2A activity at any time in culture. In contrast to insulin, IGF-I had no effect on PP-1 activity at any time in culture, but significantly increased PP-2A activity in day 5 cultures. Maximal stimulation of PP-2A activity by IGF-I was observed at 10 min, with an EC50 of 5 ng/ml. These results indicate that chick forebrain neurons contain both PP-1 and PP-2A activities and that neuronal PP-1 and PP-2A activities are differentially regulated by insulin and IGF-I. We conclude that although insulin and IGF-I share many steps in signal transduction, these growth factors have distinct actions on neuronal phosphatase activity that may impact on differences in their neurotropic actions.
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PMID:Protein phosphatase-1 and -2a activities in cultured fetal chick neurons: differential regulation by insulin and insulin-like growth factor-I. 840 57

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKC alpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.
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PMID:Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. 862 5

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A). However, after repeated washing, only PP-1 remained bound to the ribosomes. The activity of ribosome-associated PP-1 (PP-1R) was partially latent and could be increased 2-3-fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type-1 catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP-1R. We have purified from a ribosomal extract a basic polypeptide (pI > or = 10.5) of 23 kDa that potently inhibited PP-1. This ribosomal inhibitor of PP-1, termed RIPP-1, was at least 30-times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP-2A, PP-2B and PP-2C). RIPP-1 was identified as a non-competitive inhibitor of PP-1 with a substrate-dependent potency. The lowest Ki (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type-1 catalytic subunit, RIPP-1 also converted the catalytic subunit in a time-dependent manner (t 1/2 = 45 min at 25 degrees C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP-1. We propose that RIPP-1 accounts, at least in part, for the latency of PP-1R.
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PMID:Characterization of a ribosomal inhibitory polypeptide of protein phosphatase-1 from rat liver. 870 6

The protein phosphatase encoded by coliphage lambda (PPlambda) was found to be the equivalent of the minimal catalytic core of serine/threonine protein phosphatases (PP) by biochemical and mutational criteria. Bacterially expressed truncated versions of PP1 and PP5 phosphatases, representing the catalytic cores homologous to PPlambda, exhibited potent phosphatase activity. Unlike full-length PP1, but like PPlambda, the recombinant cores could use casein, p-nitrophenyl phosphate, and a wide variety of peptides as substrates and were resistant to okadaic acid, microcystin-LR, and trypsin. Mutations of His173, Asp208, or Arg221 had little effect on the activity of the PP1 core protein, indicating its closer identity with PPlambda than with full-length PP1. Terminal deletions of a few amino acids of the cores destroyed their activity, supporting their minimal nature. Analysis of PPlambda mutants suggested an influence of the substrate on metal ion binding. The minimal length of a phosphopeptide substrate of PPlambda appeared to be a phosphorylated serine/threonine flanked by 1 or 2 amino acid residues on either side, the N-terminal ones being more effective.
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PMID:Interactions between a minimal protein serine/threonine phosphatase and its phosphopeptide substrate sequence. 879 96

The adhesion molecule L-selectin is proteolytically cleaved from the surface of lymphocytes and neutrophils within minutes after stimulation by phorbol ester or calcium ionophores. In contrast to neutrophils, soluble factors have not been shown to induce down-regulation of L-selectin on lymphocytes. We therefore examined whether signals generated by interaction with cell surface receptors could deliver physiological stimuli inducing this regulatory mechanism. While cross-linking of several adhesion molecules (CD2, CD44, alpha 4-integrin, LFA-1) by antibody did not result in a significant reduction of the expression of L-selectin, antibodies against CD45 and Thy-1.2, both involved in the regulation of lymphocyte activation, induced loss of cell surface L-selectin within minutes, even at 4 degrees C, by shedding into the supernatant. Cross-linking of these molecules was shown to be essential, but Fc interactions or adherent cells were not required. A similar response, albeit less effective, was found after cross-linking of CD3. Interestingly, initiation of shedding only occurred in the presence of cell-cell contact, pointing to a second, as yet unknown, signal required. Loss of L-selectin induced by CD45 cross-linking is followed by a rapid re-expression of the molecule upon incubation at 37 degrees C. This reaction is also dependent on specific triggering signals as rapid re-expression was not observed after removal of L-selectin by trypsin. The data indicate that the protein phosphatase CD45 as well as the TCR complex itself in combination with a further, as yet unknown, cell-cell contact-dependent stimulus have a regulatory role in the dynamic control of L-selectin expression in lymphocytes.
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PMID:CD45-mediated signals can trigger shedding of lymphocyte L-selectin. 913 16

The motility of demembranated fowl spermatozoa was vigorous at 30 degrees C in the presence of ATP, but decreased markedly following the addition of recombinant protein phosphatase type 1 (PP-1) supplemented with Mn2+. This inhibition was not restored by the addition of cAMP, within the range 1-1000 microM, but instantly restored by the addition of 50 ng/ml trypsin. Phosphorylation of demembranated fowl sperm proteins during incubation with [gamma-32P]ATP at 30 degrees C was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A marked difference in phosphorylation status was observed in approximately 116, 86, 79, 50 and 29-kDa proteins. These proteins were dephosphorylated in the presence of PP-1 and Mn2+ compared with those in control samples. These results suggest that PP-1-mediated dephosphorylation of some of these proteins of the axoneme and/or accessory cytoskeletal components of fowl spermatozoa may be involved in the inhibition of motility.
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PMID:Regulation of fowl sperm flagellar motility by protein phosphatase type 1 and its relationship with dephosphorylation of axonemal and/or accessory cytoskeletal proteins. 919 45

We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.
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PMID:Cell adhesion to fibronectin regulates membrane lipid biosynthesis through 5'-AMP-activated protein kinase. 923 31

Although the calcium/calmodulin-regulated protein phosphatase calcineurin has been shown to play a role in a number of intracellular processes, relatively few of the downstream phosphoproteins that are dephosphorylated by this enzyme in cells have been described. Calcineurin was previously shown to play a role in amylase secretion by rat pancreatic acinar cells and to specifically dephosphorylate a 24-kDa cytosolic protein. The present study describes the purification and characterization of this novel phosphoprotein, termed CRHSP-24 (calcium-regulated heat-stable protein with a molecular mass of 24 kDa). Microgram quantities of CRHSP-24 were purified from a large-scale rat pancreas preparation in a procedure involving heat and acid precipitation, anion-exchange chromatography, preparative electrophoresis, electroelution, and two-dimensional electrophoresis. Internal amino acid sequence was obtained from two peptides following trypsin digestion and high pressure liquid chromatography. Both sequences matched with 100% identity nucleotide sequences of expressed sequence tags from human placenta and rat PC-12 cells. Two CRHSP-24 transcripts of 0.7 and 2. 9 kilobases were detected in multiple rat tissues by Northern analysis, whereas a single 24-kDa protein was observed by Western blotting. The CRHSP-24 protein is 147 amino acids in length, is composed of nearly 14% proline, and is phosphorylated entirely on serine residues. Western analysis and 32P metabolic labeling of acini revealed CRHSP-24 to be maximally phosphorylated in control cells and to undergo a rapid sustained dephosphorylation on at least 3 serine residues in response to calcium-mobilizing stimuli. Dephosphorylation of CRHSP-24 was completely inhibited by pretreatment of acini with cyclosporin A or FK506. Furthermore, the inhibitory effects of FK506 were blocked by excess rapamycin. The ubiquitous expression of CRHSP-24 in rat tissues suggests that this novel calcineurin substrate plays a common role in calcium-mediated signal transduction.
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PMID:Purification and characterization of a novel physiological substrate for calcineurin in mammalian cells. 971 5

Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 fused to glutathione S-transferase. The complex was inactive and required treatment with Co2+ and trypsin for maximal activity. The specific activity toward phosphorylase a was about 1.8 units/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM, respectively. The complex could be activated by glycogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the activity seen with Co2+ and trypsin. Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian protein phosphatase 1. The R73C mutant phosphatase from the glycogen-deficient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 fusion, had a specific activity toward phosphorylase a of 0.9 unit/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 13. 1, 113, and 0.37 nM, respectively. The R73C mutation slightly decreases the specific activity and sensitivity to inhibitors, suggesting that changes in biochemical properties may affect glycogen levels. However, the modest changes are consistent with our previous proposal (E. M. Reimann et al., 1993, Adv. Protein Phosphatases 7,173-182) and with the results of Stuart et al. (1994, Mol. Cell. Biol. 14, 896-905) that the mutation may selectively alter the interaction of Glc7p with regulatory proteins.
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PMID:Purification and characterization of type 1 protein phosphatase from Saccharomyces cerevisiae: effect of the R73C mutation. 972 Nov 83


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