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)

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50 = 0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.
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PMID:Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle. 923 62

CPI17, a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory activity is potentiated by protein kinase C and its related enzymes [Eto, M. et al. (1997) FEBS Lett. 410, 356-360]. In order to identify its physiological target in smooth muscle, the myofibrillar extract from porcine aorta media was analyzed by affinity chromatography on CPI17-conjugated Sepharose. The binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated myosin (p-myosin) and phosphorylase-a were bound to the resin and could be eluted with 0.5 M NaCl. The IC50 values of thiophosphorylated CPI17 toward phosphatases bound to the resin were in the range of 0.5-3 nM, as expected for the PP1 holoenzymes sensitive to CPI17. The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chromatography on Mono Q, which suggested multiple functions of CPI17 as a mediator of the protein kinase C-related signal transduction pathway in aorta smooth muscle. The major activity toward p-myosin was identified as the myofibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) was consistent with that of PP1M from chicken gizzard and porcine bladder. The purified PP1M was completely inhibited by phosphorylated and thiophosphorylated CPI17. Kinetic analysis showed mixed inhibition of PP1M by CPI17 (Ki = 1.9 nM and Ki' = 5.1 nM). The concentration of CPI17 in aorta smooth muscle cells was estimated to be at least 0. 3 microM from the result of Western analysis. This concentration appears to be sufficient to suppress the in situ PP1M in aorta smooth muscle, and PP1M is thus identified as a target of CPI17 in vascular smooth muscle.
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PMID:Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle. 999 Jan 34

cDNAs encoding KEPI, a novel protein kinase C (PKC)-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatase (PP1), were identified. They were found among morphine-regulated brain mRNAs identified using subtracted differential display techniques. Full-length rat, mouse, and human cDNA and genomic sequences were elucidated with library screening and data base searching. Rat, mouse, and human KEPI cDNAs encode 164-165 amino acid proteins with calculated isoelectric points of 5.2. Each species' amino acid sequence contains consensus sequences for phosphorylation by PKC (KVT(72)VK), protein kinase A (RKLS(154)), and casein kinase II (S(43)SRE, S(120)EEE). Multiple KEPI N-terminal myristoylation consensus sites provide potential regions for membrane anchoring. Subcellular fractionation and Western analyses revealed that most KEPI immunoreactivity was associated with P2 and P3 membrane-enriched fractions and little in cytosolic fractions. 2.6-kb KEPI mRNAs were detected in brain, especially in the cerebral cortex and hippocampus, and in heart and skeletal muscle. Brain KEPI mRNA was up-regulated by both acute and chronic morphine treatments. The human KEPI gene contains four exons extending over more than 100 kb of genomic sequence on 6q24-q25, near the mu opiate receptor gene. These sequences displayed sufficient homology with the porcine PP1 inhibitor CPI-17 that we asked whether KEPI could share the ability of CPI-17 to modulate PP1 activity in a PKC-dependent fashion. Recombinant mouse KEPI is phosphorylated by PKC with a K(m) of 2.6 microm and a t(1/2) of 20 min. Phospho-KEPI inhibits PP1alpha with an IC(50) of 2.7 nm, a potency more than 600-fold greater than that displayed by unphosphorylated KEPI. Neither phospho- nor dephospho-KEPI inhibits protein phosphatase 2A. Up-regulation of KEPI expression by morphine, an agonist at PKC-regulating G-protein-coupled mu receptors, provides a novel signaling paradigm in which the half-lives of serine/threonine phosphorylation events can be influenced by activities at G(i)/G(o)-coupled receptors that modulate KEPI expression, KEPI phosphorylation, and KEPI regulation of PP1 activity.
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PMID:KEPI, a PKC-dependent protein phosphatase 1 inhibitor regulated by morphine. 1181 71

Three novel cDNAs encoding putative protein phosphatase-1 inhibitory protein (PPP1R14A) were isolated from human, mouse and rat. The human, mouse and rat PPP1R14A proteins were all of 147 amino acids sharing about 90% sequence identity to porcine CPI17, an inhibitor of protein phosphatase-1 holoenzyme. The protein sequence 33RHARVTVK40, which is important for the inhibitory potency of porcine CPI17, was conserved in mammalian PPP1R14A. Northern blot analysis showed that human PPP1R14A was highly expressed as a 600 bp transcript in heart, prostate, testis, ovary, colon, small intestine and pancreas, lowly in brain, placenta, skeletal muscle and spleen and lung. The distribution of mouse and rat PPP1R14A was more specific and different from that of human PPP1R14A, abundant in lung, moderate/abundant in testis, moderate in brain and low in heart. In addition, we mapped human PPP1R14A to chromosome 19q13.13-q13.2 by radiation hybrid mapping, and determined that the human PPP1R14A gene spanned a 5.1-kb region and consisted of four exons and three introns.
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PMID:Identification of human, mouse and rat PPP1R14A, protein phosphatase-1 inhibitor subunit 14A, & mapping human PPP1R14A to chromosome 19q13.13-q13.2. 1193 93

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.
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PMID:Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue. 1259 64

CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.
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PMID:Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17. 1260 30

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.
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PMID:Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases. 1280 74

Protein phosphorylation regulates many fundamental processes and protein phosphatase-1 (PP1) is a major phosphatase that determines the levels of Ser/Thr phosphorylation. Regulatory subunits and inhibitor phosphoproteins control PP1 activity. PHI-1 is a member of a family of PP1 inhibitor phosphoproteins that was discovered based on sequence similarity to the known inhibitor CPI-17. To learn more about PHI-1 we determined the tissue distribution of PHI-1 in embryonic and adult tissues, and examined its cellular localization by immunohistochemistry. In the embryo PHI-1 appeared first in the heart at E10, and by E15 it was detected in multiple tissues. Expression in adult tissues was strikingly different, with PHI-1 detected primarily in smooth muscles in the intestine, blood vessels, and male and female genitourinary tracts. PHI-1 also was highly expressed in the endothelial layer of blood vessels. Both PHI-1 and CPI-17 are expressed predominantly in adult smooth muscles. Whereas CPI-17 staining was diffuse PHI-1 was concentrated along the cell membrane in distinct foci, detected by confocal and electron microscopy. The common tissue distribution but different cellular localization of PHI-1 and CPI-17 suggest distinctive physiological roles for these two PP1 inhibitors.
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PMID:Juxtamembrane localization of the protein phosphatase-1 inhibitor protein PHI-1 in smooth muscle cells. 1508 73

Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogen-targeting subunit (G(M)) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1.PP1C.P-CPI-17, leading to an increase in smooth muscle contraction.
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PMID:Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. 1518 67

We analyzed the signaling pathways initiated by endothelin receptors ETA and ETB in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC20) was mediated additively by ETA and ETB receptors and initiated by Galphaq-, Ca2+/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ETA receptors via a pathway involving sequential activation of Galpha13, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr696 and phosphorylation of MLC20. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ETB-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ETB antagonist BQ-788, but not the ETA antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ETA-dependent phosphorylation of MYPT1. In contrast, ETB initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17.
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PMID:Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB. 1547 16


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