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)

Interferon (IFN) regulatory factor 1 (IRF-1) and IRF-2 are structurally similar but functionally distinct transcription factors that bind to the positive regulatory domains I and III (PRDI/III) within the human IFN-beta promoter. To begin structure-function analysis of IRF-1 and IRF-2, the regulatory potential of carboxyl-terminal deletion mutants was analyzed by co-transfection studies in human cells and was correlated with DNA binding capacity. Transcriptional repression by IRF-2 was contained within the first 125 amino-terminal amino acids and correlated directly with IRF-2 DNA binding; deletion to a protein of 100 amino acids resulted in loss of repression and IRF-2 DNA binding. Thus, the carboxyl terminus appears dispensible for trans-repression. Hybrid constructs which fuse the DNA binding domain of IRF-1 and IRF-2 to the trans-activation domain of NF-kappa B p65 were also generated; both IRF-1/p65 and IRF-2/p65 chimeras were strong transcriptional activators. IRF-2-mediated repression was also dominant over trans-activation by these fusion proteins. The trans-activation region of IRF-1 resides in the carboxyl terminus, primarily carboxyl-terminal to amino acid 250. Mutation of three potential casein kinase II phosphorylation sites within the IRF carboxyl terminus failed to identify an essential site that contributes to IRF-1 trans-activation potential.
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PMID:Mutational analysis of interferon (IFN) regulatory factors 1 and 2. Effects on the induction of IFN-beta gene expression. 802 Dec 62

The gene encoding the 105-kDa protein (p105) precursor of the p50 subunit of transcription factor NF-kappa B also encodes a p70 I kappa B protein, I kappa B gamma, which is identical to the C-terminal 607 amino acids of p105. Here we show that alternative RNA splicing generates I kappa B gamma isoforms with properties different from those of p70. One 63-kDa isoform, termed I kappa B gamma-1, which lacks 59 amino acids C-terminal to ankyrin repeat 7, has a novel 35-amino acid C terminus encoded by an alternative reading frame of the p105 gene. A 55-kDa isoform, I kappa B gamma-2, lacks the 190 C-terminal amino acids of p70I kappa B gamma. In contrast to p70I kappa B gamma, which is a cytoplasmic protein, I kappa B gamma-1 is found in both the cytoplasm and nucleus, whereas I kappa B gamma-2 is predominantly nuclear. The I kappa B gamma isoforms also display differences in specificity and affinity for Rel/NF-kappa B proteins. While p70I kappa B gamma inhibits p50-, p65-, and c-Rel-mediated transactivation and/or DNA binding, both I kappa B gamma-1 and I kappa B gamma-2 are specific for p50 and have different affinities for this subunit. The absence in I kappa B gamma-1 and I kappa B gamma-2 of a protein kinase A site whose phosphorylation modulates p70I kappa B gamma inhibitory activity suggests that alternative RNA splicing may be used to generate I kappa B gamma isoforms that respond differently to intracellular signals.
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PMID:Alternative splicing of RNA transcripts encoded by the murine p105 NF-kappa B gene generates I kappa B gamma isoforms with different inhibitory activities. 818 15

The predominant inducible form of the NF-kappa B transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits, termed p65 and p50. Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers, p65 stimulates kappa B-directed transcription, whereas p50 functions as a kappa B-specific repressor. While authentic p50 homodimers (previously termed KBF1) have been detected in nuclear extracts from nontransfected cells, experimental evidence supporting the existence of p65 homodimers in vivo was lacking. We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells. As with the prototypical NF-kappa B p50-p65 heterodimer, this novel p65 homodimeric form of NF-kappa B is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation. Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A (RRPS; amino acids 273 to 276), both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association. In contrast, mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with I kappa B alpha. These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for I kappa B alpha recognition of p65. Taken together with prior in vivo transcription studies, these results suggest that the biological activities of p65 and p50 homodimers are independently regulated, thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-kappa B/Rel-directed gene expression.
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PMID:A novel NF-kappa B complex containing p65 homodimers: implications for transcriptional control at the level of subunit dimerization. 824 97

The p50 subunit of NF-kappa B is derived from the amino terminus of a 105 kilodalton precursor. The p105 carboxyl terminus, which contains ankyrin-like repeats, a feature of I kappa B molecules, regulates the cytoplasmic retention of p105 and inhibits DNA binding by the precursor. Here, we describe an I kappa B protein identical to the carboxyl-terminal region of p105. Probes spanning the COOH terminus but not the rel homology domain of p105 hybridize to a distinct 2.6-kilobase mRNA expressed in a wide range of murine tissues. The nucleotide sequence of complementary DNA clones for this transcript, in vitro translation, and immune precipitation of metabolically labeled cell lysates establish that it encodes a 70 kilodalton protein that corresponds to the COOH-terminal 607 amino acids of p105. p70 suppresses p65 and p75c-rel mediated transactivation of reporter genes under the control of NF-kappa B elements and in vitro can prevent DNA binding of p50 and p75c-rel homodimers to NF-kappa B sites. The ability of p70 to stably associate with p49 and p65 in vitro, but not inhibit DNA binding by these proteins, suggests that the specific inhibitory properties of this I kappa B may reflect its relative affinity for different rel targets. p70 phosphorylated by protein kinase A fails to inhibit DNA binding by p50 or the c-rel protein, and sequencing of radiolabeled p70 tryptic phosphopeptides establishes that protein kinase A phosphorylates serine residue 576 of p70. This finding suggests that the inhibitory activity of p70 can be regulated by signaling via the adenylate cyclase pathway.
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PMID:The activity of a 70 kilodalton I kappa B molecule identical to the carboxyl terminus of the p105 NF-kappa B precursor is modulated by protein kinase A. 839 3

Addition of mitogenic growth factors to quiescent cells triggers complex signal transduction cascades that result in the reprogramming of gene expression and entry into the cell cycle. We have found that an oncogenic variant of the c-Raf-1 protein kinase stimulated the expression of promoters containing NF-kappa B binding sites. In situ immunofluorescence analysis revealed elevated nuclear levels of the p65 subunit of NF-kappa B in v-raf-transformed NIH 3T3 cells. Incubation of HeLa cell cytoplasmic extracts with a purified recombinant glutathione S-transferase-raf fusion protein in the presence of ATP released active NF-kappa B that could be detected by electrophoretic gel mobility shift assay. Coincubation of purified recombinant I kappa B and glutathione S-transferase-raf in the presence of ATP resulted in the phosphorylation of I kappa B. Coexpression of GAL4 (activation domain)-I kappa B and GAL4 (DNA-binding domain)-raf fusion proteins in yeast resulted in stimulation of a GAL4-responsive reporter gene, indicating that I kappa B and Raf interact physically in vivo. These results indicate that the Raf-1 kinase functions in signal transduction in part by activating the NF-kappa B transcription factor by phosphorylating I kappa B in the cytoplasmic I kappa B-NF-kappa B complex to release active NF-kappa B.
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PMID:Raf-1 protein kinase activates the NF-kappa B transcription factor by dissociating the cytoplasmic NF-kappa B-I kappa B complex. 841 86

p65 (synaptotagmin), an abundant synaptic vesicle protein, has been implicated in the processes of vesicle docking and fusion. To characterize further the properties of this important neuronal protein, we have investigated its phosphorylation in vitro. Immunoprecipitation of p65 results in coprecipitation of a protein kinase that phosphorylates p65 as well as syntaxin, a plasma membrane protein that interacts with p65. p65 is phosphorylated on a threonine residue (Thr-128) within the cytoplasmic domain near the transmembrane region. The coprecipitating protein kinase was identified as casein kinase II based on its catalytic properties, the sequence surrounding Thr-128, and Western blot analysis of the anti-p65 immunoprecipitates. Affinity chromatography utilizing bacterially expressed fragments of p65 demonstrated that casein kinase II interacts with a domain of p65 distinct from the phosphorylation site. In a synaptic vesicle fraction, the phosphorylation of p65 is stimulated by sphingosine and by detergent solubilization, suggesting that p65 phosphorylation may be subject to regulatory processes.
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PMID:Casein kinase II phosphorylates the synaptic vesicle protein p65. 846 45

In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-kappa B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-kappa B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the I kappa B-alpha inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-kappa B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on I kappa B-alpha, as well as the possible involvement of known signalling pathways in pX-induced NF-kappa B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the I kappa B-alpha-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of I kappa B-alpha in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-kappa B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-kappa B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-kappa B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-kappa B activation by an as yet unidentified Raf-1-independent pathway.
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PMID:Hepatitis B virus pX activates NF-kappa B-dependent transcription through a Raf-independent pathway. 852 86

Effector-target cell conjugate formation is an essential step during lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Protein phosphorylation changes in human LAKs after contact with NK-resistant (LAK-sensitive) tumors were examined by two-dimensional SDS-PAGE. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets led to increased phosphorylation of two M(r) 65,000 LAK proteins, pp65a and pp65b, with isoelectric points of 5.1 and 5.2, respectively. Phosphorylation of both substrates was initiated between 1 and 5 min after coincubation with tumor targets. Contact between LAKs and targets was required for p65 phosphorylation because soluble tumor factors failed to induce phosphorylation. Normal peripheral blood lymphocyte targets, which are bound very poorly by LAKs and are resistant to killing, failed to induce LAK p65 phosphorylation. The broad protein kinase inhibitor staurosporine inhibited phosphorylation of pp65a and pp65b, supporting the hypothesis that activation of a LAK protein kinase leads to p65 phosphorylation. Cross-linking of CD16 (Fc gamma RIIIA), which mediates antibody-dependent cellular cytotoxicity in LAKs, also led to increased pp65a and pp65b phosphorylation. Collectively, these data provide correlative evidence that p65 phosphorylation may be involved in the cytolytic function of LAKs.
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PMID:Tumor target binding induces phosphorylation of two M(r) 65,000 lymphokine-activated killer proteins. 854 52

Myotrophin is a soluble-12 kilodalton protein isolated from hypertrophied spontaneously hypertensive rat and dilated cardiomyopathic human hearts. We have recently cloned the gene coding for myotrophin and expressed it in Escherichia coli. In the present study, the expression of myotrophin gene was analyzed, and at least seven transcripts have been detected in rat heart and in other tissues. We have further analyzed the primary structure of myotrophin protein and identified significant new structural and functional domains. Our analysis revealed that one of the ankyrin repeats of myotrophin is highly homologous specifically to those of myotrophin is highly homologous specifically to those of I kappa B alpha/rel ankyrin repeats. In addition, putative consensus phosphorylation sites for protein kinase C and casein kinase II, which were observed in I kappa B alpha proteins, were identified in myotrophin. To verify the significance of these homologies, kappa B gel shift assays were performed with Jurkat T cell nuclear extract proteins and the recombinant myotrophin. Results of these assays indicate that the recombinant myotrophin has the ability to interact with NF-kappa B/rel proteins as revealed by the formation of ternary protein-DNA complexes. While myotrophin-specific antibodies inhibited the formation of these complexes, rel-specific p50 and p65 antibodies supershifted these complexes. Thus, these results clearly indicate that the myotrophin protein to be a unique rel/NF-kappa B interacting protein.
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PMID:Cardiac myotrophin exhibits rel/NF-kappa B interacting activity in vitro. 857 59

The phosphoprotein I kappa B alpha exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-kappa B (p50-p65). In response to specific cellular stimulation, I kappa B alpha is further phosphorylated and subsequently degraded, allowing NF-kappa B to translocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first performed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 42 kDa were identified in cellular extracts from Jurkat T and U937 promonocytic cell lines. Specific inhibitors and immunodepletion studies identified the I kappa B alpha kinase activities as those of the alpha and alpha' subunits of casein kinase II (CKII). Immunoprecipitation studies demonstrated that CKII and I kappa B alpha physically associate in vivo. Moreover, phosphopeptide maps of I kappa B alpha phosphorylated in vitro by cellular extracts and in vivo in resting Jurkat T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Sequence analysis revealed that purified CKII and the kinase activity within cell extracts phosphorylated I kappa B alpha at its C terminus at S-283, S-288, S-293, and T-291. The functional role of CKII was tested in an in vitro I kappa B alpha degradation assay with extracts from uninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal and enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associates with I kappa B alpha in vivo, induces multisite (serine/threonine) phosphorylation, and is required for the basal and HIV-induced degradation of I kappa B alpha in vitro.
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PMID:Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation. 862 92


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