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)

Phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2alpha) is one of the best-characterized mechanisms for downregulating protein synthesis in mammalian cells in response to various stress conditions. In Drosophila, such a regulatory mechanism has not been elucidated. We report the molecular cloning and characterization of DGCN2, a Drosophila eIF-2alpha kinase related to yeast GCN2 protein kinase. DGCN2 contains all of the 12 catalytic subdomains characteristic of eukaryotic Ser/Thr protein kinases and the conserved sequence of eIF-2alpha kinases in subdomain V. A large insert of 94 amino acids, which is characteristic of eIF-2alpha kinases, is also present between subdomains IV and V. It is particularly notable that DGCN2 possesses an amino acid sequence related to class II aminoacyl-tRNA synthetases, a unique feature of yeast GCN2 protein kinase. DGCN2 expression is developmentally regulated. During embryogenesis, DGCN2 mRNA is dynamically expressed in several tissues. Interestingly, at later stages this expression becomes restricted to a few cells of the central nervous system. Affinity-purified antibodies, raised against a synthetic peptide based on the predicted DGCN2 sequence, specifically immunoprecipitated an eIF-2alpha kinase activity and recognized an approximately 175 kDa phosphoprotein in Western blots of Drosophila embryo extracts.
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PMID:Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2alpha (eIF-2alpha) kinase from Drosophila melanogaster. Homology To yeast GCN2 protein kinase. 913 6

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.
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PMID:Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2. 923 5

We determined that ribosomes of seedling roots of maize (Zea mays L.) contain the acidic phosphoproteins (P-proteins) known to form a flexible lateral stalk structure of the 60S subunit of eukaryotic ribosomes. The P-protein stalk, composed of P0, P1, and P2, interacts with elongation factors, mRNA, and tRNA during translation. Acidic proteins of 13 to 15.5 kD were released as a complex from ribosomes with 0.4 M NH4Cl/50% ethanol. Protein and cDNA sequence analysis confirmed that maize ribosomes contain one type of P1, two types of P2, and a fourth and novel P1/P2-type protein. This novel P-protein, designated P3, has the conserved C terminus of P1 and P2. P1, P2, and P3 are similar in deduced mass (11.4-12.2 kD) and isoelectric point (4.1-4.3). A 35.5- to 36-kD acidic protein was released at low levels from ribosomes with 1.0 M NH4Cl/50% ethanol and identified as P0. Labeling of roots with [32P]inorganic phosphate confirmed the in vivo phosphorylation of the P-proteins. Flooding caused dynamic changes in the P-protein complex, which affected the potential of ribosome-associated kinases and casein kinase II to phosphorylate the P-proteins. We discuss possible alterations of the ribosomal P-protein complex and consider that these changes may be involved in the selective translation of mRNA in flooded roots.
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PMID:Acidic phosphoprotein complex of the 60S ribosomal subunit of maize seedling roots. Components and changes in response to flooding. 927 49

The highly conserved protein kinase casein kinase II (CKII) is required for efficient Pol III transcription of the tRNA and 5S rRNA genes in Saccharomyces cerevisiae. Using purified factors from wild-type cells to complement transcription extracts from a conditional lethal mutant of CKII we show that TFIIIB is the CKII-responsive component of the Pol III transcription machinery. Dephosphorylation of TFIIIB eliminated its ability to complement CKII-depleted extract, and a single TFIIIB subunit, the TATA-binding protein (TBP), is a preferred substrate of CKII in vitro. Recombinant TBP purified from Escherichia coli is phosphorylated efficiently by CKII and, in the presence of a limiting amount of CKII, is able to substantially rescue transcription in CKII-deficient extract. Our results establish that TBP is a key component of the pathway linking CKII activity and Pol III transcription and suggest that TBP is the target of a CKII-mediated regulatory mechanism that can modulate Pol III transcription.
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PMID:Casein kinase II regulation of yeast TFIIIB is mediated by the TATA-binding protein. 935 48

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.
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PMID:Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway. 937 15

The first step in elongation requires two different activities; elongation factor (EF)-1alpha transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1betagammadelta catalyzes exchange of GDP on EF-1alpha with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the beta, gamma, and delta subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant beta subunit migrates as a dimer and the gamma subunit as a trimer upon gel filtration, whereas the delta subunit forms a large aggregate. Complexes of betagamma, gammadelta and betagammadelta were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between beta and delta. The activity of the recombinant subunits was determined with native EF-1alpha by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant beta and delta alone stimulated the rate of elongation by 10-fold, with a ratio of 5alpha:2beta or delta. The betagammadelta complex stimulated EF-1alpha activity up to 10-fold with a ratio of 20alpha to 1betagammadelta. Phosphorylation of the beta and delta subunits alone or in betagammadelta by protein kinase CKII had no effect on the rate of elongation.
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PMID:Recombinant subunits of mammalian elongation factor 1 expressed in Escherichia coli. Subunit interactions, elongation activity, and phosphorylation by protein kinase CKII. 940 20

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the alpha subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].
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PMID:Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity. 949 85

In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a phi C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant allows the accumulation of a threshold level of the RedD protein.
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PMID:A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket. 953 42

In proliferating cells the turnover rate of proteins responsible for regulation of the cell cycle progression, namely cyclins and inhibitors of the cyclin-dependent kinases (CDKs) and phosphatases, is rapid and their cellular level is modulated at the transcriptional, translational and/or degradation (via proteasome pathway) stages. Inhibition of proteasome function results in accumulation of rapidly turning over proteins and, thus, causes an imbalance of the cell cycle regulatory components, and loss of their regulatory function. Indeed, it has been shown that proteasome inhibitors perturb the cell cycle progression. Onconase, a novel RNase which has anti-tumor activity and is in clinical trials, has previously been shown to suppress protein synthesis, presumably by degradation of intracellular RNA, preferentially tRNA. By interfering with regulation of expression of cyclins and/or CDK-inhibitors, onconase also may induce the imbalance of these proteins and potentiate the effect of proteasome inhibitors. In the present study, we observed that the combinations of onconase with peptide-aldehyde inhibitors of calpain and proteasome such as the N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) and the N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), but not N-acetyl-leucinyl-leucinyl-methioninal (LLM), were synergistic in suppressing cell proliferation and inducing apoptosis in three human tumor cell lines: A-549 lung adenocarcinoma, DU-145 prostatic carcinoma, and MDA-MB-231 breast carcinoma. The observed cytotoxicity may also be a result of prevention of the induction of the 'survival' genes by the nuclear factor kappaB (NFkappaB) by onconase and proteasome inhibitors. The data indicate that such combinations should be further tested as potential anti-cancer regimens.
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PMID:Enhanced in vitro cytotoxicity and cytostasis of the combination of onconase with a proteasome inhibitor. 973 89

We have investigated the molecular basis of the requirement for protein kinase CK2 in nuclear transcription in Saccharomyces cerevisiae. In vivo and in vitro analysis has demonstrated that CK2 is required for efficient transcription of the tRNA and 55 rRNA genes by RNA polymerase III. This suggests that a component of the pol III transcription machinery is regulated by CK2. We tested this possibility by a biochemical complementation approach in which components of the pol III transcription machinery from wild type cells were tested for their ability to rescue transcription in extract from a conditionally CK2-deficient mutant. We found that pol III transcription initiation factor IIIB (TFIIIB) fully restores transcription in CK2-deficient extract. Further in vitro studies revealed that TFIIIB must be phosphorylated to be active, that a single subunit of wild type TFIIIB, the TATA binding protein (TBP), is efficiently phosphorylated by CK2, and that recombinant TBP and a limiting amount of CK2 rescues transcription in CK2-deficient extract. We conclude that TBP is the physiological target of CK2 among the components of the pol III transcription machinery. The implications of this result are discussed in the context of previous data concerning the regulation of TFIIIB.
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PMID:A review of progress towards elucidating the role of protein kinase CK2 in polymerase III transcription: regulation of the TATA binding protein. 1009 3


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