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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human p68 protein, an SV40 large T related antigen, is an RNA dependent ATPase and RNA helicase. It belongs to a new large and highly conserved gene family, the DEAD box proteins, whose members are involved in a variety of processes requiring manipulation of RNA secondary structure such as translation and splicing. Multiple DEAD box genes are present in S.cerevisiae, but only one has previously been described in E.coli. Low stringency screening of an E.coli genomic library with a p68 cDNA probe led to the identification of dbpA, a new E.coli DEAD box gene located at 29.6 minutes on the W3110 chromosome. We report here the nucleotide and deduced amino acid sequences of the gene. We have overexpressed dbpA from its own promoter on a high copy number plasmid and identified the gene product as a approximately 50 kD protein by immunoblotting with an anti-DEAD antibody.
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PMID:Identification of a putative RNA helicase in E.coli. 221 14

Human p68 RNA helicase is a nuclear RNA-dependent ATPase that belongs to a family of putative helicases known as the DEAD box proteins. These proteins have been implicated in aspects of RNA function including translation initiation, splicing, and ribosome assembly in a variety of organisms ranging from Escherichia coli to humans. While members of this family are believed to function in the manipulation of RNA secondary structure, little is known about the regulation of these enzymes. By immunological methods and sequence comparison, we have found that p68 possesses a region of sequence similarity to the conserved protein kinase C phosphorylation site and calmodulin binding domain (also known as the IQ domain) of the neural-specific proteins neuromodulin (GAP-43) and neurogranin (RC3). We report that p68 is phosphorylated by protein kinase C in vitro and binds calmodulin in a Ca(2+)-dependent manner. Both phosphorylation and calmodulin binding inhibited p68 ATPase activity, suggesting that the RNA unwinding activity of p68 may be regulated by dual Ca2+ signal transduction pathways through its IQ domain.
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PMID:Regulation of p68 RNA helicase by calmodulin and protein kinase C. 752 83

Members of a large family of proteins, called the DEAD box family, are ribonucleic acid binding proteins with ATPase activity. Recent investigations into the developmentally and cell type-specific expression patterns of one family member, p68 RNA helicase, suggest that this protein might play a role in organ differentiation and/or maturation, and that its expression is subject to complex regulation.
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PMID:Dead box for the living. 966 98

We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.
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PMID:A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase. 1045 30

p68 RNA helicase, a nuclear RNA helicase, was identified 2 decades ago. The protein plays very important roles in cell development and organ maturation. However, the biological functions and enzymology of p68 RNA helicase are not well characterized. We report the expression and purification of recombinant p68 RNA helicase in a bacterial system. The recombinant p68 is an ATP-dependent RNA helicase. ATPase assays demonstrated that double-stranded RNA (dsRNA) is much more effective than single-stranded RNA in stimulating ATP hydrolysis by the recombinant protein. Consistently, RNA-binding assays showed that p68 RNA helicase binds single-stranded RNA weakly in an ATP-dependent manner. On the other hand, the recombinant protein has very high affinity for dsRNA. Binding of the protein to dsRNA is ATP-independent. The data indicate that p68 may directly target dsRNA as its natural substrate. Interestingly, the recombinant p68 RNA helicase unwinds dsRNA in both 3' --> 5' and 5' --> 3' directions. This is the second example of a Asp-Glu-Ala-Asp (DEAD) box RNA helicase that unwinds RNA duplexes in a bi-directional manner.
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PMID:The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase. 1182 73

We previously reported ATPase, RNA unwinding, and RNA-binding activities of recombinant p68 RNA helicase that was expressed in Escherichia coli. Huang et al. The recombinant protein bound both single-stranded (ss) and double-stranded (ds) RNAs. To further characterize the substrate RNA binding by p68 RNA helicase, we expressed and purified the recombinant N-terminal and C-terminal domains of the protein. RNA-binding property and protein phosphorylation of the recombinant domains of p68 were analyzed. Our data demonstrated that the C-terminal domain of p68 RNA helicase bound ssRNA. More interestingly, the C-terminal domain was a target of protein kinase C (PKC). Phosphorylation of the C-terminal domain of p68 abolished its RNA binding. Based on our observations, we propose that the C-terminal domain is an RNA substrate binding site for p68. The protein phosphorylation by PKC regulates the RNA binding of p68 RNA helicase, which consequently controls the enzymatic activities of the protein.
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PMID:Phosphorylation of p68 RNA helicase regulates RNA binding by the C-terminal domain of the protein. 1473 53

We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the bacterially expressed p68 RNA helicase is phosphorylated on tyrosine, serine, and threonine residues. Our data demonstrated that phosphorylations on the recombinant p68 RNA helicase affect the enzymatic activities of the protein. This is the first observation that recombinant protein expressed in bacteria Escherichia coli is phosphorylated at multiple residues by bacterial endogenous protein kinases. Our observations suggest an important mechanism in controlling the function of p68 RNA helicase by signal transduction pathways.
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PMID:Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues. 1513 10

P68 nuclear RNA helicase is essential for normal cell growth. The protein plays a very important role in cell development and proliferation. However, the molecular mechanism by which the p68 functions in cell developmental program is not clear. We previously observed that bacterially expressed his-p68 was phosphorylated at multiple sites including serine/threonine and tyrosine [L. Yang, Z.R. Liu, Protein Expr. Purif., 35: 327]. Here we report that p68 RNA helicase is phosphorylated at tyrosine residue(s) in HeLa cells. Phosphorylation of p68 at threonine or tyrosine residues responds differently to tumor necrosis factor alpha (TNF-alpha)induced cell signal. Kinase inhibition and in vitro kinase assays demonstrate that p68 RNA helicase is a cellular target of p38 MAP kinase. Phosphorylation of p68 affects the ATPase and RNA unwinding activities of the protein. In addition, we demonstrate here that phosphorylation of p68 RNA helicase controls the function of the protein in the pre-mRNA splicing process. Interestingly, phosphorylation at different amino acid residues exhibits different regulatory effects. The data suggest that function(s) of p68 RNA helicase may be subjected to the regulation of multiple cell signal pathways.
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PMID:Signaling to the DEAD box--regulation of DEAD-box p68 RNA helicase by protein phosphorylations. 1592 48

We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5' splice site (5'ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5'ss, while the mutants still interacted with the U1-5'ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5'ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.
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PMID:ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome. 1610 97

The fundamental biology and the biochemical processes at different developmental stages of the malaria parasite Plasmodium falciparum have not been explored in detail. As a step toward understanding the various mechanisms engaged in nucleic acid metabolism of this pathogen, particularly the essential enzymes involved in nucleic acid unwinding, recently, we have reported the isolation of the first P. falciparum DEAD-box DNA helicase 60 (PfDH60), which contained striking homology with p68 protein [Pradhan A, Chauhan VS, Tuteja R. A novel 'DEAD-box' DNA helicase from Plasmodium falciparum is homologous to p68. Mol Biochem Parasitol 2005;140:55-60]. In this study, we show novel important properties of PfDH60. Immunofluorescence assay studies revealed that the peak expression of PfDH60 is mainly in the schizont stages of the development of P. falciparum, where DNA replication is active. Interestingly, this is a bipolar DNA helicase, which unwinds dsDNA in both the directions. PfDH60 can also unwind RNA-DNA and RNA-RNA duplexes. PfDH60 is phosphorylated by protein kinase C at the Ser and Thr residues. The helicase and ATPase activities of PfDH60 were stimulated after this phosphorylation. The cell-cycle dependent expression, bipolar translocation and dual nature collectively suggest that PfDH60 may be involved in the process of DNA replication and distinct cellular processes in the parasite and this study should make an important contribution in our better understanding of DNA metabolic pathways such as repair, recombination and replication.
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PMID:Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. 1616 32


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