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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.
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PMID:Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. 963 26

Phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B are critical players in cell proliferation and survival. Their downstream effector protein kinase, p70 S6 kinase, has an established role in protein translation. The mechanism by which bacterial LPS induces production of nitric oxide (NO) in murine macrophages is incompletely understood, and a role for PI 3-kinase/p70 S6 kinase pathway had not been previously investigated. In this study we demonstrate that LPS induced a fivefold activation of p70 S6 kinase and a twofold stimulation of PI 3-kinase. Pretreatment of Raw 264.7 cells with either rapamycin or Ly290042 completely blocked LPS-induced activation of p70 S6 kinase. Protein kinase B was also activated (twofold) by LPS and was only minimally affected by these inhibitors. PI 3-kinase activity was inhibited by both Ly294002 and wortmannin. The effects on NO production by these agents were strikingly different. While both rapamycin and Ly294002 resulted in almost complete inhibition of NO production, wortmannin was ineffective. Surprisingly, none of the inhibitors reduced the production of the inducible nitric oxide synthase protein (iNOS) as determined by immunoprecipitation. In vivo labeling studies revealed that the iNOS protein was phosphorylated in concordance with the production of NO. We conclude that LPS-mediated NO production occurs via a PI 3-kinase-independent, but FKBP12-rapamycin-associated protein-dependent, pathway in RAW cells by a mechanism probably involving phosphorylation of iNOS.
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PMID:Activation of phosphatidylinositol 3-kinase, protein kinase B, and p70 S6 kinases in lipopolysaccharide-stimulated Raw 264.7 cells: differential effects of rapamycin, Ly294002, and wortmannin on nitric oxide production. 986 29

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.
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PMID:Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast. 1043 10

A novel inositolphosphate-binding protein has been identified and shown to be an immunophilin. This protein, which was isolated from human erythrocyte membranes and from K562 (human erythroleukemia) cell membranes, has robust peptidylprolyl cis-trans isomerase activity that is strongly inhibited by nanomolar concentrations of FK506 or rapamycin, indicating a member of the FKBP (FK506-binding protein) class. However, unlike the cytosolic FKBP12, the isomerase activity of this membrane-associated immunophilin is strongly inhibited by nanomolar concentrations of inositol 1,4, 5-trisphosphate (IP(3)), inositol 1,3,4,5-tetrakisphosphate (IP(4)), and phosphatidylinositol 4- and 4,5-phosphates, which are suggested to be physiological ligands. The demonstration of a single 12-kD protein that binds both IP(4) or IP(3) and anti-FKBP12 provides strong support for the inositolphosphate-binding immunophilin having an apparent mass of 12 kD, and it is suggested that the protein might be called IPBP12 for 12-kD inositol phosphate binding protein. When an internal tryptic peptide derived from IPBP12 was sequenced, a sequence also present in human cytokeratin 10 was identified, suggesting a cytoskeletal localization for the immunophilin. While purifying IPBP12, it was found that it is immunoprecipitated with specific proteins that include a protein kinase and a phosphoprotein phosphatase. The latter is indicated to be phosphoprotein phosphatase 2A (PP-2A). It is suggested that immunophilins promote the assembly of multiprotein complexes that often include a protein kinase or a phosphoprotein phosphatase or both.
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PMID:An inositolphosphate-binding immunophilin, IPBP12. 1051 81

Ultraviolet radiation induces signal transduction at both early (<6 h) and late (>6 h) times after exposure. The inflammatory and immunosuppressive cytokine tumor necrosis factor alpha is induced at late times, and is induced by ultraviolet-induced DNA damage, as defects in DNA repair increase, and enhanced photoproduct repair reduces, tumor necrosis factor alpha expression. Here we show that late tumor necrosis factor alpha gene expression is sensitive to rapamycin, implicating FKBP12-rapamycin-associated protein, a member of the DNA protein kinase family, as a signal transducer of ultraviolet-induced DNA damage. FKBP12-rapamycin-associated protein was localized in the nucleus of keratinocytes and its level was increased following ultraviolet irradiation. Immuno- precipitated FKBP12-rapamycin-associated protein was stimulated by ultraviolet-irradiated DNA to phosphorylate p53 in vitro, and in vivo rapamycin reduced ultraviolet induction of p53 by 20%. Rapamycin further inhibited the ultraviolet-induced phosphorylation of the FKBP12-rapamycin-associated protein downstream target kinase p70S6K. In mice, topical application of rapamycin before ultraviolet exposure protected against suppression of the contact hypersensitivity that is a hallmark of ultraviolet-induced cytokine gene expression. These results demonstrate that the FKBP12-rapamycin-associated DNA protein kinase transduces the signal of ultraviolet-induced DNA damage into production of immunosuppressive cytokines at late times after ultraviolet irradiation.
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PMID:FRAP DNA-dependent protein kinase mediates a late signal transduced from ultraviolet-induced DNA damage. 1077 84

We have previously shown that interferon-alpha (IFN alpha)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12-rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN alpha signaling by serine phosphorylating IRS-1. We found that both FRAP and insulin-activated p70 S6 kinase (p70(s6k)) serine phosphorylated IRS-1 between residues 511 and 772 (IRS-1(511-772)). Importantly, only FRAP-dependent IRS-1(511-772) serine phosphorylation inhibited by 50% subsequent JAK1-dependent tyrosine phosphorylation of IRS-1. Furthermore, treatment of U266 cells with the FRAP inhibitor rapamycin increased IFN alpha-dependent tyrosine phosphorylation by twofold while reducing constitutive IRS-1 serine phosphorylation within S/T-P motifs by 80%. Taken together, these data indicate that FRAP, but not p70(s6k), is a likely physiologic IRS-1 serine kinase that negatively regulates JAK1-dependent IRS-1 tyrosine phosphorylation and suggests that FRAP may modulate IRS-dependent cytokine signaling.
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PMID:Frap-dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation. 1116 88

We have cloned and characterized a new member of the phosphatidylinositol kinase (PIK)-related kinase family. This gene, which we term human SMG-1 (hSMG-1), is orthologous to Caenorhabditis elegans SMG-1, a protein that functions in nonsense-mediated mRNA decay (NMD). cDNA sequencing revealed that hSMG-1 encodes a protein of 3031 amino acids containing a conserved kinase domain, a C-terminal domain unique to the PIK-related kinases and an FKBP12-rapamycin binding-like domain similar to that found in the PIK-related kinase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activity as measured by autophosphorylation and phosphorylation of the generic PIK-related kinase substrate PHAS-1. hSMG-1 kinase activity is inhibited by high nanomolar concentrations of wortmannin (IC(50) = 105 nm) but is not inhibited by a FKBP12-rapamycin complex. Mutation of conserved residues within the kinase domain of hSMG-1 abolishes both autophosphorylation and substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic protein kinase activity. hSMG-1 phosphorylates purified hUpf1 protein, a phosphoprotein that plays a critical role in NMD, at sites that are also phosphorylated in whole cells. Based on these data, we conclude that hSMG-1 is the human orthologue to C. elegans SMG-1. Our data indicate that hSMG-1 may function in NMD by directly phosphorylating hUpf1 protein at physiologically relevant sites.
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PMID:Cloning of a novel phosphatidylinositol kinase-related kinase: characterization of the human SMG-1 RNA surveillance protein. 1133 Dec 69

The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.
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PMID:The rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin. 1237 Feb 90

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation-contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are "leaky." RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.
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PMID:PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure. 1262 52

Defective calcium (Ca2+) signaling and impaired contractile function have been observed in skeletal muscle secondary to impaired myocardial function. However, the molecular basis for these muscle defects have not been identified. In this study, we evaluated the alterations of the ryanodine-sensitive Ca2+ release channels (RyR1) by analyzing global and local Ca2+ signaling in a rat postmyocardial infarction (PMI) model of myocardial overload. Ca2+ transients, measured with multiphoton imaging in individual fibers within a whole extensor digitorum longus (EDL) muscle, exhibited significantly reduced amplitude and a prolonged time course in PMI. Spatio-temporal properties of spontaneous Ca2+ sparks in fibers isolated from PMI EDL muscles were also significantly altered. In addition, RyR1 from PMI skeletal muscles were PKA-hyperphosphorylated and depleted of the FK506 binding protein (FKBP12). These data show that PMI skeletal muscles exhibit altered local Ca2+ signaling, associated with hyperphosphorylation of RyR1. The observed changes in Ca2+ signaling may contribute to defective excitation-contraction coupling in muscle that can contribute to the reduced exercise capacity in PMI, out of proportion to the degree of cardiac dysfunction.
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PMID:Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats. 1282 80


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