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)

Switching Saccharomyces cerevisiae from non-fermentative to fermentative growth by adding glucose to a medium with glycerol as the sole carbon source, leads to a sudden increase in the rate of ribosomal protein gene transcription. By analyzing the nutritional shift response in a variety of yeast mutants and in the presence of different drugs, evidence was obtained that: (i) no de novo protein synthesis is required for this response; (ii) protein kinase A is essential, though independent of intracellular levels of cAMP, whereas protein kinase C is not involved; (iii) proper regulation of sugar phosphorylation is essential; (iv) glycolysis is required for the long term effect of the nutritional upshift; and (v) pathways leading to glucose-induced activation differ from those leading to gene repression, probably already at the level of glucose transport.
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PMID:Nutritional upshift response of ribosomal protein gene transcription in Saccharomyces cerevisiae. 798 81

The Arabidopsis Atpk1 protein expressed in insect cells and plant cells exhibited multiple sizes consisting mainly of two doublets: p70 (68 and 70 kDa) and p85 (82 and 85 kDa). Extraction of p85 from cells required the presence of SDS, suggesting that p85 is associated with less soluble subcellular components. p70 was extracted by nonionic detergent without SDS, indicating that this form is cytoplasmic. p70 expressed in either Arabidopsis or insect cells underwent serine-specific autophosphorylation, indicating that Atpk1 is a protein-serine kinase. A point mutation (lysine 163 to arginine) in the ATP-binding site of the catalytic domain substantially diminished activity when expressed in insect cells. A 14-kDa protein (p14) was co-immunoprecipitated with p70 from insect cells expressing wild-type Atpk1 and was phosphorylated in immune complex kinase assays with Atpk1, suggesting it is a homolog of a natural substrate of Atpk1. Two plant ribosomal proteins (14 and 16 kDa) can be phosphorylated by the Atpk1 protein kinase, and we propose that Atpk1 is a novel ribosomal protein kinase. A 60-kDa form of Atpk1 derived from the insect cell-expressed p70 was more highly phosphorylated than p70 in in vitro kinase assays, suggesting a negative regulatory domain can be removed by proteolysis.
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PMID:atpk1, a novel ribosomal protein kinase gene from Arabidopsis. II. Functional and biochemical analysis of the encoded protein. 802 Dec 67

Bacteriophage T7 expresses a serine/threonine-specific protein kinase activity during infection of its host, Escherichia coli. The protein kinase (gp0.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub-optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphorylated in T7-infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electrophoretic data moreover indicate that elongation factor P is phosphorylated in T7-infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two-dimensional gel pattern, and to confirm that serine is the phosphate-accepting amino acid. The two-dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which is a significantly higher number than previous estimates. The protein kinase activities of the T7-related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7-infected cells.
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PMID:Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli. 802 76

A ribosomal protein S2 kinase was purified 6000-fold from cytoplasmic extracts of HeLa cells infected with vaccinia virus, using 80S ribosomes or 40S ribosomal subunits as a substrate. Although the preparation was not homogeneous, a 34K component was identified, the chromatographic behaviour of which correlated with enzyme activity. During its purification the ribosomal protein S2 kinase was resolved from a less abundant ribosomal protein S13 kinase, demonstrating the two to be different entities. A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity during all five chromatographic procedures employed, and we conclude that the two activities are properties of a single species. Two-dimensional gel electrophoresis demonstrated that this second substrate was the acidic ribosomal protein Sa, of isoelectric point approximately 5.2, previously shown to be phosphorylated during infection with vaccinia virus. Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approximately 5.0, which is also known to be phosphorylated in vivo. The 34K protein correlating with the catalytic activity in the most purified preparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vaccinia virus gene B1R. This and other evidence suggest strongly that the ribosomal protein S2/Sa kinase is the product of this gene.
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PMID:Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein. 811 49

Yeast ribosomal protein genes are coordinately regulated as a function of cell growth; RNA levels decrease during amino acid starvation but increase following a carbon source upshift. Binding sites for RAP1, a multifunctional transcription factor, are present in nearly all ribosomal protein genes and are associated with growth rate regulation. We show that ribosomal protein mRNA levels are increased twofold in strains that have constitutively high levels of cyclic AMP-dependent protein kinase (protein kinase A [PKA]) activity. The PKA-dependent induction requires RAP1 binding sites, and it reflects increased transcriptional activation by RAP1. Growth-regulated transcription of ribosomal protein genes strongly depends on the ability to regulate PKA activity. Cells with constitutively high PKA levels do not show the transcriptional decrease in response to amino acid starvation. Conversely, in cells with constitutively low PKA activity, ribosomal protein mRNAs levels are lower and largely uninducible upon carbon source upshift. We suggest that modulation of RAP1 transcriptional activity by PKA accounts for growth-regulated expression of ribosomal protein genes.
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PMID:Protein kinase A mediates growth-regulated expression of yeast ribosomal protein genes by modulating RAP1 transcriptional activity. 811 23

Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71-95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.
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PMID:Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library. 812 53

We have expressed the rat brain Ca2+/calmodulin (CaM)-dependent protein kinase type IV in insect cells. The recombinant enzyme is produced as a single polypeptide that migrates on SDS-polyacrylamide gel electrophoresis at 61 kDa. Recombinant CaM kinase IV undergoes slow CaM-dependent autophosphorylation. The autophosphorylation of CaM kinase IV occurs on serine residues but is not accompanied by the generation of a CaM-independent activity, as previously reported for the cerebellar enzyme. Comparison of peptide and protein phosphorylation by the recombinant CaM kinase IV and the cerebellar enzyme showed differences in their catalytic activities. The deduced primary sequence of CaM kinase IV contained a domain, 315Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys323, also found in the regulatory domain of CaM kinase II alpha (residues 293-300). Truncation of CaM kinase IV at Leu313 (at a position analogous to Leu290 in CaM kinase II alpha) generated a fully active, CaM-independent enzyme. This truncated enzyme no longer bound CaM. These data confirm that CaM kinase IV demonstrates intrasteric regulation by an autoinhibitory domain and provides insight into a potentially common mechanism for the regulation of the CaM-dependent multifunctional protein kinases. A number of synthetic peptides were examined for their phosphorylation by both CaM kinase II and IV. These studies showed that several peptides derived from phospholamban were preferential substrates for CaM kinase II whereas a peptide derived from S6 ribosomal protein was selectively phosphorylated by CaM kinase IV. Kinetic analysis of several peptide substrates suggests that while both CaM kinase II and IV recognize the sequence motif represented by R-X-X-T/S, other structural features are also involved in defining the unique substrate specificity of CaM kinase IV.
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PMID:Biochemical characterization of the multifunctional Ca2+/calmodulin-dependent protein kinase type IV expressed in insect cells. 825 36

Synthetic peptides have been used to define specificity determinants and to distinguish reactivities of numerous protein kinases and phosphoprotein phosphatases. Direct analysis of peptide phosphorylation is most often determined using P81 phosphocellulose paper to separate modified peptide and unreacted [gamma-32P]ATP; however phosphopeptide dephosphorylation is usually determined by extraction and quantitation of phosphomolybdate complexes or ion exchange chromatography. We describe here the adaptation of the rapid, direct P81 paper protein kinase assay for the determination of phosphopeptide dephosphorylation. The S6-21 peptide (AKRRRLSSLRASTSKSESSQK), which is derived from the multiphosphorylated carboxyl terminal domain of the S6 ribosomal protein, was phosphorylated by a human placenta S6 kinase and dephosphorylation by purified phosphoprotein phosphatase type 1 in the presence of a variety of buffers, and inhibitors/activators was determined using the new assay. Results comparable to those obtained with the ion-exchange chromatography were obtained, and the assay was significantly less expensive, more rapid, and more accurate than methods previously used to quantitate phosphopeptide dephosphorylation.
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PMID:Protein phosphatase assay using a modification of the P81 paper protein kinase assay procedure. 838 82

By using site-directed mutagenesis and chemical analysis of phosphopeptides, a unique phosphorylation site has been shown at serine 73 in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal protein YP1 beta (L44'). The mutation in this position prevents in vitro phosphorylation by protein kinases that modify the wild-type polypeptide. The unphosphorylatable mutated protein is unable to bind to the ribosomes and to rescue the growth deficiency of yeast strains in which the corresponding original gene is inactivated by gene disruption. Sequencing of tryptic phosphopeptides has shown that acidic proteins YP1 alpha and YP2 alpha (L44) are also phosphorylated at positions near the carboxyl end. These results contrast with the data indicating that in the highly homologous protein YP2 beta, phosphorylation takes place at serine 19, close to the amino terminus. The results show that phosphorylation is definitely required for the biological activity of these ribosomal proteins. However, the differences in the phosphorylation sites suggest that the effect of this modification is not the same in all of them, confirming the heterologous role of these peculiar ribosomal components. In fact, the different context of the modification sites in the four polypeptides suggests the existence of more than one protein kinase specific for this set of proteins.
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PMID:The activity-controlling phosphorylation site is not the same in the four acidic ribosomal proteins from Saccharomyces cerevisiae. 842 20

We have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV of Saccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp. NO422 and NO425 correspond to the split ribosomal protein genes encoding S16A and rp28, respectively, NO450 displays a striking similarity with serine/threonine protein kinase genes, in particular with STE20, and therefore may encode a novel member of this protein family. NO453 is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known. NO530 encodes the plasma membrane protein Mid1p and NO533 corresponds to the gene coding for a 40 kDa subunit of replication factor C. The remaining ORFs show weak or no homology with proteins in the data bases.
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PMID:Sequence analysis of a 30 kb DNA segment from yeast chromosome XIV carrying a ribosomal protein gene cluster, the genes encoding a plasma membrane protein and a subunit of replication factor C, and a novel putative serine/threonine protein kinase gene. 855 2


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