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)

When mouse 3T3-F442A preadipocyte fibroblasts reach confluence in the appropriate culture medium, their growth is arrested, and the cells undergo terminal differentiation to adipocytes. Two proteins that may be involved in this process are interferon and the interferon-induced double-stranded RNA (dsRNA)-dependent protein kinase (DAI). In 3T3-F442A cells, interferon and DAI are transiently expressed with a maximum level of active kinase appearing at confluence. Interestingly, the level of active DAI was found to be low when cells were maintained under conditions nonpermissive for differentiation. This reduction in DAI was at least partly because of the presence of elevated levels of a specific inhibitor of DAI, termed dRF, which appeared to be a reversible inhibitor of the autophosphorylation (activation) of DAI. In the present study, the mechanism of action of dRF was investigated. Photocross-linking experiments indicated that dRF prevented the binding of ATP to DAI. Since the binding of ATP to DAI is dsRNA-dependent, we examined the effect of dRF on the binding of dsRNA to the kinase using RNA mobility shift assays. dRF was found to prevent the formation of DAI-dsRNA complexes without a direct effect on the dsRNA. This suggests that dRF exerts its effect through an interaction with DAI.
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PMID:Mechanism of action of a cellular inhibitor of the dsRNA-dependent protein kinase from 3T3-F442A cells. 135 90

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.
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PMID:Interactions between double-stranded RNA regulators and the protein kinase DAI. 135 46

P68 is a protein kinase expressed by eukaryotic cells, which is inducible by alpha interferon, and is believed to be an important factor in the regulation of viral and cellular protein synthesis. We have previously reported on a monoclonal antibody, TJ4C4, which is able to specifically detect p68 in formalin-fixed, paraffin-embedded tissue. Because of its important role in regulating cellular protein synthesis, we hypothesized that p68 expression would vary among lung neoplasms with level of differentiation and degree of biosynthetic activity. A total of 246 untreated primary pulmonary and pleural neoplasms were studied. The frequency and relative intensity of p68 expression was determined by light microscopic evaluation of ABC immunoperoxidase stained specimens. All categories of tumors studied demonstrated a spectrum of p68 expression. Expression of p68 correlated well with degree of differentiation in squamous cell carcinomas (SQCC) and acinar adenocarcinomas (AAC). Papillary adenocarcinoma (PAC) and bronchioalveolar carcinoma (BAC) expressed low levels of p68, despite their well differentiated appearance. Expression of the antigen in large cell carcinoma (LCC) was higher than that seen in either poorly differentiated AAC or SQCC. Neuroendocrine tumors generally showed low levels of p68 expression with the intermediate variant of small cell carcinoma expressing higher levels of p68 than the classic "oat cell" form (SCC). Carcinoid tumors expressed higher levels of p68 than did atypical carcinoid tumors. Mesotheliomas showed weak expression of p68, limited primarily to areas of glandular differentiation in the epithelioid form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of the protein kinase p-68 recognized by the monoclonal antibody TJ4C4 in human lung neoplasms. 135 15

The interferon (IFN)-inducible double-stranded (ds) RNA-activated protein kinase (p68 kinase) is a physiologically important enzyme that regulates the rate of cellular and viral protein synthesis by phosphorylating and thereby inactivating the peptide chain initiation factor 2. We have generated a partial cDNA clone, which probably represents the murine p68 kinase, by reverse transcription-polymerase chain reaction (RT-PCR) using sequence information of the human p68 kinase. The 725-bp cDNA clone encoded the carboxyl-terminal 238 amino acid residues of the mouse kinase. It has 67% overall identity with the corresponding region of the human kinase. All the protein kinase catalytic domains are conserved in the mouse protein. Moreover, there are additional stretches of residues that are totally conserved between the two proteins. The functional equivalence of the two proteins was tested by constructing a chimeric cDNA that encoded a protein whose amino-terminal 364 residues were of human origin and carboxyl-terminal 187 residues were of mouse origin. The chimeric protein was as efficient as the human p68 kinase in binding to the dsRNA, autophosphorylating and phosphorylating exogenous substrate.
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PMID:Construction and expression of an enzymatically active human-mouse chimeric double-stranded RNA-dependent protein kinase. 135 89

The P68 protein kinase (referred to as P68 based on its M(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsRNAs. The kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elF-2), leading to potential limitations in functional elF-2 and decreases in protein synthesis initiation. To further delineate the molecular mechanisms underlying kinase regulation, we attempted to express the P68 protein kinase in insect cells using a baculovirus vector. Repeated efforts to isolate recombinant baculoviruses containing a wild-type kinase failed, whereas recombinants expressing a nonfunctional kinase with a catalytic domain II mutation were readily isolated. When used to infect Spodoptera frugiperda cells, the recombinant virus expressed the exogenous mutant protein at almost 5-10% of the total proteins synthesized. We then purified the kinase by immunoaffinity chromatography to raise monospecific antiserum which recognized not only the human native wild-type P68, but also kinase homologues in murine, bovine, and monkey cells as determined by immunoblot and immunoprecipitation analysis. Fortunately, kinase function also could be assayed using this antibody since the human and nonhuman kinase homologues, present in immunoprecipitates, were autophosphorylated and phosphorylated the natural substrate, elF-2 alpha. Further, this antiserum recognized epitopes throughout the molecule including the amino and carboxyl termini in contrast to the available monoclonal antibody. In vitro assays using the polyclonal antibody revealed the importance of the amino terminus, especially amino acids 1-97, in the binding of the kinase to viral RNA activators and inhibitors. Finally, we determined that the P68 amino terminus was both necessary and sufficient for binding dsRNA as we were able to transfer dsRNA-binding properties to a reporter gene product previously unable to bind RNA.
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PMID:Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase. 136 Jan 80

The protein kinase DAI, the double-stranded RNA-activated inhibitor of translation, is an essential component of the interferon-induced cellular antiviral response. The enzyme is regulated by the binding of activator and inhibitor RNAs. We synthesized DAI in vitro and located its RNA-binding domain within the amino-terminal 171 residues. This domain contains two copies of an RNA-binding motif characterized by a high density of basic amino acids, by the presence of conserved residues, and by a probable alpha-helical structure. Deletion of either of the two motifs prevents the binding of dsRNA, but their relative positions can be exchanged, suggesting that they cooperate to interact with dsRNA. Clustered point mutations within the RNA-binding motifs and duplications of the individual motifs indicate that the first copy of the motif plays the more important role. Mutations that impair binding have similar effects on the binding of double-stranded RNAs of various lengths and of adenovirus VA RNAI, implying that discrimination between activator and inhibitory RNAs takes place subsequent to RNA binding.
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PMID:Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. 136 13

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.
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PMID:Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. 137 53

A molecular cDNA clone (KIN27) was isolated that encodes a novel variant of the catalytic subunit C alpha of the human cyclic AMP-dependent protein kinase (PKA). Analysis of PKA genomic sequence data revealed that the KIN27 variant transcripts likely result from alternative splice-site selection of the C alpha gene transcripts. The KIN27 cDNA has therefore been designated C alpha 2. RNA amplification by polymerase chain reaction revealed that KIN27 C alpha 2 splice-site variant RNA and PKA C alpha RNA were both expressed in the amnion U and HeLa human cell lines. C alpha RNA was about 3- to 5-fold more abundant than C alpha 2 RNA. Interferon treatment decreased the steady-state amount of the C alpha 2 RNA relative to C alpha RNA. These results suggest that alternative splicing may contribute to the structural heterogeneity of C subunits expressed in human cells and that interferon may affect this process.
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PMID:Mechanism of interferon action. cDNA structure and regulation of a novel splice-site variant of the catalytic subunit of human protein kinase A from interferon-treated human cells. 137 29

The work described in this report suggests the existence of two biochemically distinguishable forms of the interferon-inducible, double-stranded RNA-dependent protein kinase. Kinase isolated from the cytosolic fraction (S-100) and the ribosome salt wash fraction of interferon-treated cells differed in their chromatographic properties. S-100 kinase eluted from a gel filtration column with M(r) = 140,000-160,000 and was predominantly anionic in nature, whereas ribosomal kinase eluted with M(r) = 66,000 and was predominantly cationic in nature. Purified preparations of S-100 kinase contained the M(r) = 66,000 subunit, P1, as the only polypeptide present in stoichiometric amounts, and thus the S-100 kinase appears to be a dimer of P1 subunits. Dimerization of the S-100 kinase was dependent on the phosphorylation state of the enzyme. Kinase isolated from S-100 was partially phosphorylated. Dephosphorylation of the S-100 kinase by treatment with alkaline phosphatase resulted in a monomeric form of the enzyme with biochemical characteristics similar to that of the ribosome salt wash kinase.
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PMID:Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr = 66,000 subunits. 137 30

The P68 protein kinase is a serine/threonine kinase induced by interferon treatment and activated by double-stranded RNAs (dsRNAs). Once activated, the kinase phosphorylates its natural substrate, the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently purified from influenza virus-infected cells a P68 kinase inhibitor, found to be a 58-kDa cellular protein. We have now investigated the mechanisms by which the 58-kDa inhibitor regulates P68 kinase activity and how the inhibitor itself is controlled. The 58-kDa inhibitor did not function by degrading or sequestering the dsRNA activator of P68 but could repress phosphorylation of eIF-2 alpha by an already activated protein kinase. Utilizing antibody prepared against a 58-kDa-specific peptide, we showed that the 58-kDa proteins from infected and uninfected cells were present in equivalent amounts. Although kinase inhibitory activity could not be detected in crude uninfected cell extracts, ammonium sulfate treatment unmasked this activity and allowed purification of the cellular inhibitor with identical chromatographic properties as that from influenza virus-infected cells. Finally, we have identified and partially purified a specific inhibitor of the 58-kDa protein which we refer to as an "anti-inhibitor." Based on these data, we present a model depicting the complex regulation of the interferon-induced protein kinase in eukaryotic cells.
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PMID:Characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsRNA-activated protein kinase. 137 38


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