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)

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15-20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.
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PMID:Characterization of the reversible phosphorylation and activation of ERK8. 1633 13

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.
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PMID:A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. 1673 47

PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all alpha-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity.
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PMID:Crystal structure of the PP2A phosphatase activator: implications for its PP2A-specific PPIase activity. 1688 30

WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na(+)-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K(+)-driven cotransporters KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine-rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 on the K(+)-Cl(-) cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K(+)-Cl(-) cotransporters KCC1, KCC3, and KCC4 under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. In contrast, three different WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K(+)-Cl(-) cotransporters expressed in the kidney.
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PMID:WNK4 kinase is a negative regulator of K+-Cl- cotransporters. 1718 32

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.
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PMID:Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells. 1722 12

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus laevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase.
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PMID:Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation. 1726 45

Paclitaxel (Ptx) is an antitumoural drug that inhibits microtubule dynamics, causes G2/M arrest and induces cell death. 2-D PAGE and MALDI-TOF-MS analysis of HeLa cells extracts revealed that Ptx up-regulates a form of the eukaryotic elongation factor 1Bgamma (eEF1Bgamma) and down-regulates another one. This event, linked to the lack of Ptx effect over eEF1Bgamma mRNA or protein levels suggested a PTM of this elongation factor. Further 2-D PAGE analysis followed by a phosphospecific staining with PRO-Q Diamond showed the staining of the Ptx up-regulated form only. Moreover, this Ptx up-regulated form of eEF1Bgamma disappears upon treatment with protein phosphatase. Thus, we demonstrate that human eEF1Bgamma phosphorylation is regulated by Ptx.
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PMID:Phosphorylation of human eukaryotic elongation factor 1Bgamma is regulated by paclitaxel. 1770 94

Phosphorylation and dephosphorylation of neurotransmitter receptors represent an important mechanism to regulate synaptic signal transduction. Here, we identified PNUTS, a targeting subunit of protein phosphatase 1 (PP1) as a new binding partner of GABA(C) receptors. In the mammalian retina, PNUTS is co-expressed with GABA(C) receptors and PP1 in bipolar cells. PNUTS and PP1 were detected in membrane protein preparations of the retina and precipitate with GABA(C) receptor specific antibodies. Furthermore, PNUTS shuttles from the nucleus to the membrane in cells co-expressing GABA(C) receptors. We show simultaneous binding of PP1 and GABA(C) receptors to different domains of PNUTS, demonstrating that PNUTS cross-links PP1 and GABA(C) receptors. Finally, modeling studies showed that the PP1 docking motif of PNUTS fits into the binding pocket on the enzyme surface, despite a C-terminal adjacent proline. We suggest that PNUTS targets PP1 to synaptic sites, acting as a temporary bridge between the phosphatase and GABA(C) receptors.
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PMID:PNUTS forms a trimeric protein complex with GABA(C) receptors and protein phosphatase 1. 1832 84

Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. We used a multifaceted strategy including 32P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from 32P-labeled synaptosomes, phosphopeptides were isolated from proteolytic digests using TiO2 chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC separation and two-dimensional tryptic mapping of 32P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest 32P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of 32P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate 32P during the 1-h labeling period. Multiple phosphosites from amphI-co-migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, alpha/beta-adducin, and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.
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PMID:The in vivo phosphorylation sites in multiple isoforms of amphiphysin I from rat brain nerve terminals. 1834 31

Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.
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PMID:Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1. 1847 60


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