title: Rat leucine-rich protein binds and activates the promoter of the beta isoform of Ca2+/calmodulin-dependent protein kinase II gene.
abstract: We previously found the neuronal cell-type specific promoter and binding partner of the beta isoform of Ca(2+)/calmodulin-dependent protein kinase II (beta CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of beta isoform of Ca(2+)/calmodulin-dependent protein kinase II of rat brain. Mol. Brain Res. 94, 35-47]. In the present study, we purified a protein that binds specifically a promoter region of beta CaM kinase II gene from a nuclear extract of the rat cerebellum using DEAE-cellulose column chromatography, ammonium sulfate fractionation, gel filtration and polyacrylamide gel electrophoresis. The purified protein was identified as rat leucine-rich protein 157 (rLRP157) using tandem mass spectrometry. Then, we prepared its cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly(A)(+)RNA of rat cerebellum. The rLRP157 cDNA was introduced into mouse neuroblastomaxrat glioma hybrid NG108-15 cells, and cells stably expressing rLRP157 (NG/LRP cells) were isolated. Binding of rLRP157 with the promoter sequence was confirmed by electrophoretic mobility shift assay using nuclear extract of NG/LRP cells. A luciferase reporter gene containing a promoter of beta CaM kinase II was transiently expressed in NG/LRP cells. Under the conditions, the promoter activity was enhanced about 2.6-fold in NG/LRP cells as compared with wild-type cells. The expression of rLRP157 mRNA was paralleled with that of beta CaM kinase II in the adult and embryo rat brain detected by in situ hybridization. Nuclear localization of rLRP157 was confirmed using GFP-rLRP157 fusion protein investigated under a confocal microscope. These results indicate that rLRP157 is one of the proteins binding to, and regulating the activity of, the promoter of beta CaM kinase II.
authors.affiliation: Department of Biochemistry, Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1, Tokushima 770-8505, Japan.
authors.forename: Nagahiro
authors.initials: N
authors.lastname: Ochiai
authors.forename: Shuji
authors.initials: S
authors.lastname: Masumoto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sakagami
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Yoshimura
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamauchi
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000201:enzymology / Q000254:growth & development; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics*; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D002531:Cerebellum / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006825:Hybridomas; D008156:Luciferases / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D053719:Tandem Mass Spectrometry; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000235:genetics
chemical_list: C497030:Lrpprc protein, rat; D016601:RNA-Binding Proteins; D008156:Luciferases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517672:Camk2b protein, mouse; C517676:Camk2b protein, rat