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)

A unique and highly conserved structural feature of approximately 90-kDa ribosomal S6 kinase (p90rsk or RSK) is the presence of two non-identical kinase domains. To explore the mechanism of RSK activation, a cloned human RSK cDNA (RSK3) was used to generate and characterize several site-directed RSK mutants; K91A (N-Lys, NH2-terminal ATP-binding mutant), K444A (C-Lys, COOH-terminal ATP-binding mutant), N/C-Lys (double ATP-binding mutant) T570A (C-Thr, mutant of the putative MAPK phosphorylation site in subdomain VIII of the C-domain), S218A (N-Ser, mutant of the corresponding NH2-terminal residue). Epitope-tagged RSKs were expressed in transfected COS cells followed by immunoprecipitation with or without prior in vivo epidermal growth factor stimulation. Kinase activity (S6 peptide) of N/C-Lys and N-Lys was ablated (and partially impaired with N-Ser). In contrast, both C-Lys and C-Thr retained high levels of kinase activity and were capable of responding to stimulation. C-Lys also retained partial kinase activity toward other substrates (c-Fos, S40 ribosomes, protein phosphatase 1 G-subunit, histones, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)) whereas N-Lys did not. The isolated NH2-and COOH-terminal domains were also expressed; the C-domain was inactive, whereas the N-domain retained partial activity. Relative to wild-type, both N-Lys and C-Lys (as well as N-Ser and C-Thr) underwent partial in vitro autophosphorylation that was further stimulated by EGF protein tyrosine phosphatase. We conclude that 1) the NH2-terminal RSK kinase domain mediates substrate phosphorylation; 2) both domains contribute to autophosphorylation; 3) the putative MAPK phosphorylation site is not required for growth factor-stimulated autophosphorylation or kinase activation.
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PMID:Divergent functional roles for p90rsk kinase domains. 764 38

Na(+)/H(+) exchanger isoform-1 (NHE1), the ubiquitous form of the Na(+)/H(+) exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P)(703)) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P)(703) (RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P)(703) exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 +/- 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 +/- 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and +2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P)(703). GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the base-line rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 +/- 0.02 versus 0.48 +/- 0.06 mmol of H(+)/min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3.
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PMID:14-3-3 Binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange. 1127 64

Compound 5 (Cpd 5) or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is an inhibitor of protein phosphatase Cdc25A and causes persistent activation of extracellular signal-regulated kinase (ERK) and cell growth inhibition. To study the mechanism(s) by which persistent ERK phosphorylation might induce cell growth inhibition, we used Cpd 5 as a tool to examine its effects on the activity of CREB (cAMP response element-binding protein) transcription factor in Hep3B human hepatoma cells. We found that CREB activity, including its DNA binding ability and phosphorylation on residue Ser-133, was strongly inhibited by Cpd 5, followed by suppression of CRE-mediated transcription of cyclin D1 and Bcl-2 genes. Cpd 5-mediated suppression of CREB phosphorylation and transcriptional activity was antagonized by mitogen-activated protein kinase kinase inhibitors PD 98059 and U-0126, implying that this inhibition of CREB activity was regulated at least in part by the ERK pathway. The phosphorylation of ribosomal S6 kinase (pp90(RSK)), a CREB kinase in response to mitogen stimulation, was also found to be inhibited by Cpd 5 action. This inhibition of pp90(RSK) phosphorylation is likely the result of its increased association with CREB-binding protein (CBP), which subsequently caused inhibition of CREB phosphorylation and activity. To support the hypothesis that Cpd 5 effects on Cdc25A inhibition with subsequent ERK activation could cause CREB inhibition, we examined the effects of Cdc25A inhibition without the use of Cpd 5. Hep3B cells were transfected with C430S Cdc25A mutant, and ERK was found to be phosphorylated in a constitutively activated manner, which was accompanied by decreased CREB phosphorylation and increased recruitment of CBP to pp90(RSK). These data provide evidence that CBP.RSK complex formation in response to persistent ERK phosphorylation by Cpd 5 down-regulates CREB activity, leading to inhibition of both cAMP response element-mediated gene expression and cell growth.
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PMID:Persistent ERK phosphorylation negatively regulates cAMP response element-binding protein (CREB) activity via recruitment of CREB-binding protein to pp90RSK. 1254 Aug 38

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cdelta (protein phosphatase 2Cdelta). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cd is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cdelta (H154D) (His154-->Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1-71 of PP2Cd and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cd associates with RSK1-RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cd in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.
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PMID:p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cdelta. 1520 6

Integration of protein kinases into transcription activation complexes influences the magnitude of gene expression. The nuclear factor of activated T cells (NFAT) group of proteins are critical transcription factors that direct gene expression in immune and nonimmune cells. A balance of phosphotransferase activity is necessary for optimal NFAT activation. Activation of NFAT requires dephosphorylation by the calcium-mediated calcineurin phosphatase to promote NFAT nuclear accumulation, and the Ras-activated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase, which targets NFAT partners, to potentiate transcription. Whether protein kinases operate on NFAT and contribute positively to transcription activation is not clear. Here, we coupled DNA affinity isolation with in-gel kinase assays to avidly pull down the activated NFAT and identify its associated protein kinases. We demonstrate that p90 ribosomal S6 kinase (RSK) is recruited to the NFAT-DNA transcription complex upon activation. The formation of RSK-NFATc4-DNA transcription complex is also apparent upon adipogenesis. Bound RSK phosphorylates Ser(676) and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser(676) is also targeted by the ERK MAP kinase, which interacts with NFAT at a distinct region than RSK. Thus, integration of the ERK/RSK signaling pathway provides a mechanism to modulate NFATc4 transcription activity.
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PMID:Recruitment of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway to the NFATc4 transcription activation complex. 1565 20

In this study we compared the content and phosphorylation levels of several molecules believed to regulate muscle hypertrophy and fiber type changes in the extensor digitorum longus (EDL), soleus, diaphragm, and heart of adult (6 months), aged (30 months), and very aged (36 months) Fischer 344 x Brown Norway rats. With aging, the mass of the EDL and soleus decreased significantly (approximately 38% and approximately 36%, respectively), the diaphragm's mass remained unchanged while the mass of the heart increased (approximately 35%). Western blotting demonstrated that calcineurin (CnA), the 70-kDa ribosomal S6 kinase (p70(S6k)), glycogen synthase kinase-3beta (GSK-3beta), and the phosphorylated forms of GSK-3beta and p70(S6k) (p-GSK-3beta(Ser9) and p-p70(S6kThr389)) were regulated differently with aging and between muscle types. Total p70(S6k), GSK-3beta, and p-GSK-3beta(Ser9) decreased in the aged-atrophic EDL and soleus while p-p70(S6kThr389) increased. Although total p70(S6k) content diminished in the continuously active diaphragm, phosphorylation of p70(S6k )remained unchanged. Conversely, the expression of GSK-3beta and p-GSK-3beta(Ser9) increased in the diaphragm. With aging, the amount of p-p70(S6kThr389) decreased approximately 56% in the heart while p-GSK-3beta( Ser9) increased approximately 193%. Interestingly, CnA content remained unchanged in the diaphragm, increased approximately 204% in the EDL, and decreased approximately 30% and approximately 65% with aging in the soleus and heart, respectively. These results indicate remarkable differences in the regulation of molecules thought to govern protein synthesis and changes in contractile protein expression.
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PMID:Regulation of p70S6k, GSK-3beta, and calcineurin in rat striated muscle during aging. 1604 21

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.
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PMID:Effect of branched-chain amino acids on muscle atrophy in cancer cachexia. 1762 10

Haloperidol, a classical antipsychotic drug, affects the extracellular signal-regulated kinase (ERK) pathway in the brain. However, findings are inconsistent and the mechanism by which haloperidol regulates ERK is poorly understood. Therefore, we examined the ERK pathway and the related protein phosphatase 2A (PP2A) in detail after haloperidol administration. Haloperidol (0.5 and 1 mg/kg) induced biphasic changes in the phosphorylation level of mitogen-activated protein kinase kinase (MEK), ERK, and p90 ribosomal S6 kinase (p90RSK) without changing Raf-1 phosphorylation. Fifteen minutes after haloperidol administration, MEK-ERK-p90RSK phosphorylation increased, whilst PP2A activity decreased. At 60 min, the reverse was observed and the binding of PP2A to MEK and ERK increased. Higher dosages of haloperidol (2 and 4 mg/kg), affected neither MEK-ERK-p90RSK phosphorylation nor PP2A activity. Accordingly, PP2A regulates acute dose- and time-dependent changes in MEK-ERK-p90RSK phosphorylation after haloperidol treatment. These findings suggest the involvement of a dephosphorylating mechanism in the acute action of haloperidol.
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PMID:Haloperidol regulates the phosphorylation level of the MEK-ERK-p90RSK signal pathway via protein phosphatase 2A in the rat frontal cortex. 1827 21

Sarcopenia is the loss of muscle mass and strength which occurs with aging. Whether the molecular basis of sarcopenia differs with muscle type and across sex is not well understood. Here we examine how aging affects the regulation of protein kinase B (Akt), the mammalian target of rapamycin (mTOR), AMP activated kinase (AMPK), p70 ribosomal S6 kinase (p70s6k), S6 ribosomal protein (rps6) and calcineurin (CaN) in the slow soleus and fast extensor digitorum longus (EDL) muscles of 6- (adult), 30- (aged), and 36-month (very aged) male and 6- (adult), 26- (aged), and 30-month (very aged) female Fischer 344xBrown Norway (F344BN) rats. In male animals, soleus and EDL muscle to body weight ratios decreased steadily with age while in the females, losses remained unchanged after 26 months. These age-related changes in the degree of muscle atrophy across sex were associated with differences in the regulation of Akt, mTOR, and p70s6k in the slow-twitch soleus and the regulation of AMPK, 4EBP1, p70s6k and rpS6 in the fast-twitch EDL. Irrespective of muscle type, aging in both the genders was associated with increased calcineurin expression. Taken together, these data suggest that indices of protein synthesis and muscle adaptation are regulated differently with aging in different muscle types and sex.
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PMID:Effects of aging and gender on muscle mass and regulation of Akt-mTOR-p70s6k related signaling in the F344BN rat model. 2015 66

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.
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PMID:Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1. 2123 2


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