title: The murine nucleolin protein is an inducible DNA and ATP binding protein which is readily detected in nuclear extracts of lipopolysaccharide-treated splenocytes.
abstract: A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA lambda gt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The beta-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.
authors.affiliation: Department of Biology, University of California at Los Angeles 90024-1606, USA.
authors.forename: G A
authors.initials: GA
authors.lastname: Miranda
authors.forename: I
authors.initials: I
authors.lastname: Chokler
authors.forename: R J
authors.initials: RJ
authors.lastname: Aguilera
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation; D000097796:Nucleolin
chemical_list: D000255:Adenosine Triphosphate; D000906:Antibodies; D004247:DNA; D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; D008070:Lipopolysaccharides; D008934:Mitogens; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097796:Nucleolin