THE MURINE NUCLEOLIN PROTEIN IS AN INDUCIBLE DNA AND ATP BINDING-PROTEIN WHICH IS READILY DETECTED IN NUCLEAR EXTRACTS OF LIPOPOLYSACCHARIDE-TREATED SPLENOCYTES

A 100-kDa DNA binding protein was found to be dramatically up-regulate d upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also fou nd to exhibit moderate binding specificity for the immunoglobulin isot ype switch DNA repeats. Furthermore, the induction of the 100-kDa prot ein 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 pro tein was one of the most heavily phosphorylated proteins in both lymph oid and nonlymphoid nuclear extracts. Although this in vitro phosphory lation initially appeared to be mediated by a potent nuclear kinase ac tivity, it was later determined that a significant part of the detecte d 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 is olate cDNA clones from a lymphocyte cDNA lambda gt11 expression librar y, Nucleotide sequence analysis revealed that the cloned cDNAs were id entical to the mouse nucleolin gene. The beta-galactosidase fusion pro teins (encoded by exons 3-14 of nucleolin) and a more severely truncat ed 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 bin ding 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 con served RNA binding domains of nucleolin. (C) 1995 Academic Press, Inc.