Rat leucine-rich protein binds and activates the promoter of the β isoform of Ca 2+/calmodulin-dependent protein kinase II gene

Abstract

We previously found the neuronal cell-type specific promoter and binding partner of the β isoform of Ca 2+/calmodulin-dependent protein kinase II (β CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of β 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 β 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 neuroblastoma × rat 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 β 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 β 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 β CaM kinase II.