a Novel Nuclear Matrix Protein , Associates With p 110 RB and Is Involved in Neuronal Differentiation

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain–like structure in the predicted NH 2 terminus, and a “kelch motif” in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110 RB ) is found to be associated with the nuclear matrix and overexpression of p110 RB induces neuronal differentiation, we investigated whether NRP/B is associated with p110 RB . Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110 RB during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110 RB and participates in the regulation of neuronal process formation. T he nuclear matrix is formed by an ordered and highly compartmentalized protein structure consisting of a nuclear lamina, a residual nucleolus, and an internal matrix composed of a nonchromatin fibrogranular network associated with DNA (Berezney and Coffey, 1974; Berezney, 1984, 1991; Buttyan and Olsson, 1986; Schuchard et al., 1991; Carter et al., 1993; Xing et al., 1993). The nuclear matrix has been implicated in transcription, regulation of gene expression, cell cycle, primary transcription processing, and linkages to intermediate filaments of the cytoskeleton (He et al., 1995; Loidl and Eberharter, 1995; Penman, 1995; Mancini et al., 1996). The nuclear lamins and the nuclear matrins were identified as major nuclear matrix proteins (Hakes and Berezney, 1991 a , b ; Nakayasu and Berezney, 1991). The nuclear matrix, as an underlying nuclear framework, coordinates the different processes occurring at the chromatin sites, which are under stringent cell cycle control (Loidl and Eberharter, 1995). For example, the retinoblastoma gene product (p110 RB ) is associated with the nuclear matrix in a cell cycle–dependent manner (Mittnacht and Weinberg, 1991; Mancini et al., 1994; Riley et al., 1994). Isolated nuclear matrices from synchronized cultured cells contained a significant amount of hypophosphorylated retinoblastoma protein only during the G1 period (Goodrich et al., 1991). Some p110 RB -binding proteins are themselves associated with the nuclear matrix, inThis paper is dedicated to Raphael Recanati and his family for their friendship and support for our research program. Address all correspondence to Dr. Shalom Avraham, Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, 3rd Floor, Boston, MA 02115. Tel. : (617) 667-0063. Fax: (617) 975-5240. Sequence data are available from GenBank/EMBL/DDBJ under accession number AF059611. on A uust 2, 2017 jcb.rress. org D ow nladed fom