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 effect of membrane permeable cAMP analogues on the expression of extracellular superoxide dismutase (EC-SOD) was studied in rat C6 glioma. EC-SOD is constitutively expressed but stimulation with cAMP analogues still increased the EC-SOD transcription and the secreted SOD activity. The potency to enhance EC-SOD expression is correlated with the ability of the cAMP analogue to induce cAMP-dependent differentiation in C6. The increase in EC-SOD mRNA and in secreted activity depended on the concentration of the cAMP analogues and on the cultivation time. Twenty-four hours after addition of 0.5 mM N6, O'2-dibutyryl cAMP (dbcAMP) or N6-monobutyryl cAMP (N6-mbcAMP) EC-SOD mRNA expression increased approximately twofold, while stimulation for 68 h with 0.5 mM N6-mbcAMP or 1 mM 8-Chloro cAMP (ClcAMP) and 1 mM dbcAMP enhanced the mean secreted activity/cell three- and fivefold, respectively. O'2-monobutyryl cAMP (O'2-mbcAMP) did not affect EC-SOD synthesis. The enhancement in EC-SOD activity did not require activation of protein kinase A. ATP, TGF-beta, IFN-gamma, and LPS did not affect EC-SOD synthesis. The presented data point to a cAMP-dependent pathway for the enhanced expression of EC-SOD by glial cells in brain.
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PMID:Cyclic AMP-induced differentiation increases the synthesis of extracellular superoxide dismutase in rat C6 glioma. 888 98

Human 17 beta-hydroxysteroid dehydrogenase (17-HSD) type 1 catalyzes the conversion of the low activity estrogen, estrone, into highly active estradiol, both in the gonads and in target tissues. The present study was carried out to characterize the dimerization, microheterogeneity, and phosphorylation of human 17-HSD type 1 and to evaluate the current model of hydride transfer and substrate recognition of the enzyme, based on its x-ray structure. 17-HSD type 1 is a homodimer consisting of noncovalently bound subunits, and the data in the present study indicate an exceptionally strong association between the monomers [dissociation constant (Kd) < 5 pmol/monomers liter]. Furthermore, substitutions constructed at the hydrophobic dimer interface always resulted in inactive aggregates of the protein. The enzyme was shown to be phosphorylated by protein kinase A exclusively at Ser134 only in vitro. However, in contrast to previous suggestions, phosphorylation of Ser134 was shown to play no role in the activity or microheterogeneity of human 17-HSD type 1. The presence of microheterogeneity in the recombinant enzyme also indicates that it does not result from the frequent protein polymorphism previously found for the enzyme. In line with the x-ray structure and the proposed catalytic mechanism of the enzyme, our results indicate that Ser142, Tyr155, and Lys159 are all critical for hydride transfer in human 17-HSD type 1. In contrast, the proposed interaction between His221, Glu282, and the 3-OH group of the steroid at the substrate recognition helix could not be shown to exist. Neither of these residues plays a critical role in the catalytic action of the enzyme in cultured cells.
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PMID:Characterization of structural and functional properties of human 17 beta-hydroxysteroid dehydrogenase type 1 using recombinant enzymes and site-directed mutagenesis. 899 90

A bacterial expression vector is described for investigation of protein-protein interactions. Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI. Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)). The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions. It has been shown that the product of the repressor gene with newly incorporated expressed polylinker restriction sites still functions as a repressor. Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm. Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell. Complete restoration is achieved using a host E. coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells. Human PKA has been reconstituted in the E. coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.
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PMID:A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex. 903 6

In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hydrodynamic parameters indicative of a homodimer. Chemical cross-linking studies showed that pig cGK II in intestinal membranes has a dimeric structure as well. However, after purification, cGK II was found to be partly proteolyzed into C-terminal monomeric fragments. Phosphorylation studies in rat intestinal brush borders revealed that the potency of cGMP analogs to stimulate or inhibit native cGK II in vitro (i.e. 8-(4-chlorophenylthio)-cGMP > cGMP > beta-phenyl-1,N2-etheno-8-bromo-cGMP > beta-phenyl-1,N2-etheno-cGMP and Rp-8-(4-chlorophenylthio)-cGMPs > Rp-beta-phenyl-1, N2-etheno-8-bromo-cGMPs, respectively) correlated well with their potency to stimulate or inhibit cGK II-mediated Cl- secretion across intestinal epithelium but differed strikingly from their potency to affect cGK I activity. These data show that the N terminus of cGK II is involved in dimerization and that endogenous cGK II displays a distinct activation/inhibition profile with respect to cGMP analogs, which permits a pharmacological dissection between cGK II- and cGK I-mediated physiological processes.
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PMID:Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can Be functionally distinguished from the type I isoforms. 911 39

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator that acts on a diverse set of nuclear genes required for mitochondrial respiratory function in mammalian cells. These genes encode respiratory proteins as well as components of the mitochondrial transcription, replication, and heme biosynthetic machinery. Here, we establish that NRF-1 is a phosphoprotein in vivo. Phosphorylation occurs on serine residues within a concise NH2-terminal domain with the major sites of phosphate incorporation at serines 39, 44, 46, 47, and 52. The in vivo phosphorylation pattern can be approximated in vitro by phosphorylating recombinant NRF-1 with purified casein kinase II. Phosphate incorporation at the sites utilized in vivo results in a marked stimulation of DNA binding activity which is not observed in mutated proteins lacking these sites. Pairwise expression of the wild-type protein with each of a series of truncated derivatives in transfected cells results in the formation of a dimer between wild-type and mutant forms demonstrating that a homodimer is the active binding species. Although NRF-1 can dimerize in the absence of DNA, phosphorylation does not enhance the formation of these dimers. These findings suggest that phosphorylation results in an intrinsic change in the NRF-1 dimer enhancing its ability to bind DNA.
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PMID:Serine phosphorylation within a concise amino-terminal domain in nuclear respiratory factor 1 enhances DNA binding. 922 45

Interferon-gamma (IFN-gamma) is a potent inhibitor of hematopoiesis in vitro and has been implicated in the pathophysiology of human bone marrow failure syndromes. IFN-gamma both inhibits cell cycling and induces expression of the Fas-receptor, resulting in subsequent apoptosis of hematopoietic progenitor cells. IFN regulatory factor-1 (IRF-1) mediates some of these suppressive effects by activation of downstream inducible genes, such as double-stranded RNA-activatable protein kinase and inducible nitric oxide synthase. However, under certain experimental conditions, IFN-gamma appears to stimulate proliferation of hematopoietic cells. Based on the hypothesis that IFN-gamma-receptor triggering may activate diverse signaling cascades, we designed experiments to determine which intracellular mechanisms (in addition to the IRF-1 transduction pathway) influence the biologic effects of IFN-gamma. Using antisense technique, we inhibited the IRF-1-mediated pathway in KG1a cells stimulated with IFN-gamma. In contrast to the suppressive effects of IFN-gamma observed in control cells, untreated and IFN-gamma-treated KG-1a cells that were transduced with retroviral vectors expressing IRF-1 antisense mRNA showed enhanced proliferation. The increased growth rate was associated with decreased levels of IRF-1 mRNA and protein but unchanged levels of IRF-2. We inferred that IFN-gamma could also activate a stimulatory transduction pathway that, under specific conditions, may control the cellular response to this cytokine. The family of Stat proteins is involved in signal transduction of hematopoietic growth factors. We showed that, in KG-1a cells, IFN-gamma also induced phosphorylation of Stat1 and Stat3, whereas p42 MAP kinase was phosphorylated regardless of the presence of IFN-gamma. Using electrophoresis mobility shift assays, IFN-gamma enhanced Stat1-Stat1 homodimer and Stat1-Stat3 heterodimer formation, suggesting that, in addition to inhibitory signals mediated by IRF-1, IFN-gamma may activate proliferative signals by phosphorylation of Stat1 and Stat3 proteins. The observations made in experiments with KG-1a cells were confirmed in primary hematopoietic cells. After inhibition of the IRF-1 pathway by transduction of an antisense IRF-1 retrovirus into human CD34+ cells, IFN-gamma produced an aberrant stimulatory effect on hematopoietic colony formation. Conversely, in control vector-transduced CD34+ cells, the typical inhibitory response to IFN-gamma was seen. Our results indicate that inhibitory cytokines such as IFN-gamma may exhibit diverse biologic effects depending on the intracellular balance of transcriptional regulators, in turn influenced by the activation and differentiation status of the target cells.
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PMID:Inhibition of interferon regulatory factor-1 expression results in predominance of cell growth stimulatory effects of interferon-gamma due to phosphorylation of Stat1 and Stat3. 938 91

Recent evidence demonstrates that the proto-oncogene product, Bcl-2 can protect cells from a variety of cell death-inducing stimuli. Because previous studies have demonstrated that protein kinase (PK) pathways may be involved in the regulation of cell death, we tested various PK inhibitors for their effects on cell death in a dopaminergic neuronal cell line, MN9D, as well as the potential of Bcl-2 family members and structural mutants to block this process. Cells expressing either human Bcl-2 (MN9D/Bcl-2), or neomycin (MN9D/Neo; control cells) were treated with either staurosporine (0.25-2 microM) or trifluoperazine (10-100 microM). In control MN9D/Neo cells, both reagents led to a dose-dependent cell death with morphological features of apoptosis. Overexpression of Bcl-2 rescued cells from staurosporine-induced but not trifluoperazine-induced apoptotic cell death. Cell death induced by the specific PKC inhibitor, calphostin C was also significantly attenuated in MN9D/Bcl-2 cells indicating that a PKC pathway represents one mechanism by which Bcl-2 prevents staurosporine-induced cell death. Similarly, the Bcl-2 family member, Bcl-X(L) also blocked staurosporine-induced cell death in MN9D cells whereas overexpression of Bcl-X(S) or Bax did not. Finally, staurosporine-induced cell death was still blocked by the expression of clones encoding mutations in the Bcl-2 homology domains, BH1 and BH2, as well as C-terminally truncated Bcl-2. These data suggest that in the staurosporine-mediated cell death model Bcl-2 is not heterodimerizing to related proteins through these highly conserved structural domains nor does it need to be membrane-anchored. Thus, in this paradigm, either Bcl-2 functions as a homodimer or essential sequences lie outside of the BH1 or BH2 domains.
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PMID:Regions outside of the Bcl-2 homology domains, BH1 and BH2 protect a dopaminergic neuronal cell line from staurosporine-induced cell death. 942 15

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.
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PMID:The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase. 943 Jul 29

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.
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PMID:14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove. 963 91

PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and alpha-actinin. To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed. Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library. The interaction between PKN and PCD17 was also determined by the in vitro binding analysis. PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17. PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 microM arachidonic acid. The amino-terminal region of PCD17 could form a homodimer in vitro, and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells. These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus.
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PMID:PKN interacts with a paraneoplastic cerebellar degeneration-associated antigen, which is a potential transcription factor. 963 78


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