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)

Raf is a mitogen-stimulated protein kinase that functions as a component of the signaling cascade that leads to the stimulation of mitogen-activated protein kinase. Here we show that the native structure of Raf is a large multi-subunit protein complex with an apparent mass of 300-500 kDa that interacts with Ras and the mitogen-activated protein kinase kinase Mek. Analysis of the structure of the Raf complex demonstrates that it contains a single Raf protein kinase together with the molecular chaperones hsp90 and p50. The Raf-hsp90-p50 complex was observed in starved cells and in cells activated with serum or phorbol ester. Thus, changes in complex formation with hsp90 and p50 are not required for activation of the Raf protein kinase. However, Raf activation caused by Ras was associated with the translocation of the cytoplasmic Raf-hsp90-p50 complex to the cell membrane. Significantly, it is only the membrane-bound complex that exhibits increased protein kinase activity. Thus, the Ras-activated Raf protein kinase functions as a membrane-bound multi-subunit complex.
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PMID:The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex. 812 27

The Met proto-oncogene product is a tyrosine kinase receptor whose ligand is hepatocyte growth factor (HGF). The Met protein is first synthesized in the hepatocytes as a single chain precursor, or p170MET proreceptor, and is then processed to a mature heterodimer receptor consisting of an extracellular alpha subunit (p50 alpha MET) and a transmembrane beta subunit (p 145 beta MET). The beta subunit has a protein kinase domain which is activated through phosphorylation on tyrosine residue by the binding of HGF to the receptor. In order to elucidate the function of the Met gene product in hepatic disorders, we analyzed the expression and tyrosine phosphorylation of the Met protein on regeneration and carcinogenesis of the liver. For studies on carcinogenesis we used human hepatoma tissues, and for studies on regeneration we used rat hepatectomy. Two antibodies were used for western blotting; a mouse monoclonal anti-phosphotyrosine antibody, which recognizes phosphorylated tyrosine residue in proteins, and a polyclonal rabbit anti-Met antibody, which recognizes the C-terminus of both the Met beta chains and proreceptor. To compare the amount of protein in each experiment, the results of western blotting were evaluated using an image analyzing system. In experiments involving rats with partial hepatectomy, a decreased expression of the proreceptor with a decreased amount of tyrosine phosphorylation was observed within 12 hours of hepatectomy. However, there were no significant changes of the Met beta subunit during the experiment. These data suggest that the Met proreceptor is decreased in the early stages of liver regeneration. In experiments on human samples surgically removed from 18 patients with hepatocellular carcinoma, the met proteins, p 145 beta MET and p 160 MET proreceptor, were expressed both in cancer tissues (12/18, and 10/18, respectively) and in non-cancer tissues (8/18, and 15/18, respectively). From the comparative analyses of the intensity of the signals in cancerous region against those of non-cancerous region in the 18 individual cases, it was demonstrated that expression of p 160 MET proreceptor was increased in non-cancerous region more significantly than in cancerous region (p < 0.05). On the contrary, expression of p145 beta MET was increased in cancerous region more significantly than in non-cancerous region (p < 0.05), except for a few cases of poorly differentiated carcinomas in which p 145 beta MET signal was not detected. These findings suggested that a processing pathway from the proreceptor to the mature Met receptor is amplified in carcinogenesis of the liver.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Analysis of the hepatocyte growth factor receptor in regeneration and oncogenesis of hepatocytes]. 815 55

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

A precursor, p105, for one of the subunits (p50) of the NF-kappa B transcription factor, plays an important role in inducible expression of diverse cellular genes. p105 also functions as a cytoplasmic inhibitor for NF-kappa B, and the proteolytic processing of its inhibitory C-terminal region is required for generation of active NF-kappa B. Here, it is reported that the human p105 C-terminal region is phosphorylated in vivo on Ser894 and Ser908, which are potential phosphorylation sites in vitro for proline-directed serine/threonine kinases such as cyclin-dependent kinase. Furthermore, the mutation of these in vivo phosphorylation sites retards p105 processing in vivo, suggesting that p105 processing is regulated in a phosphorylation-dependent manner.
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PMID:A role for phosphorylation in the proteolytic processing of the human NF-kappa B1 precursor. 852 73

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

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

Persistent human immunodeficiency virus (HIV) infection of human monocytes and macrophages increases I kappa B alpha degradation, resulting in the activation of NF-kappa B, a key transcription factor in the regulation of the HIV long terminal repeat. The signal transduction pathways leading to NF-kappa B activation in cells of the monocytic lineage, especially those regulated by HIV infection, and their relevance in regulating viral persistence remain unknown. Both p21ras and its downstream Raf-1 kinase participate in the transduction of signals initiated from a variety of cell surface receptors and in the regulation of transcription factors. We have studied whether the Ras-Raf pathway is functional and participates in HIV-mediated NF-kappa B activation in monocytic cells. Constitutively active p21ras (v-H-Ras) activated NF- kappa B-dependent transcription and induces the nuclear translocation of a bona fide p65/p50 heterodimer by targeting I kappa B alpha. In addition, the constitutively active form of Raf (RafBXB) also increases the NF-kappa B-dependent transcriptional activity. Because of the similarity between HIV and Ras-Raf-induced NF-kappa B activation in monocytic cells, we next tested whether HIV-induced NF-kappa B activation was mediated by the Ras-Raf signal transduction pathway. Negative dominant forms of both Ras (Ras N17) and Raf (Raf 301) decreased the HIV- but not lipopolysaccharide-dependent NF-kappa B activation in U937 cells. Moreover, Raf-1 kinase activity was greater in HIV-infected than uninfected monocytic cells in in vitro kinase assays. Altogether, these results indicate that the Ras-Raf pathway is unregulated in HIV monocytic cells and participates in the virus-induced activation of NF-kappa B.
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PMID:The Ras-Raf pathway is activated in human immunodeficiency virus-infected monocytes and particpates in the activation of NF-kappa B. 864 60


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