CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.

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Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1 −/− mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases. [BMB Reports 2021; 54(3): 164-169] BMB Rep. 2021; 54(3): 164-169 www.bmbreports.org ATP1B1 is a new binding partner of NEGR1 Yeongmi Cheon, et al.

Cancer-upregulated gene 2 (CUG2), which was named since it was originally identified as a putative oncogene up-regulated in various human cancers, was recently renamed CENP-W based on the new findings that it is a component of the centromeric complex playing a crucial role in the assembly of functional kinetochore complex during mitosis. To understand the transcriptional regulation of CENP-W, we analyzed its TATA-less promoter and identified a GC-rich putative Sp1 binding site located at -46 to -36 that was critical in CENP-W expression. Competitive electrophoretic gel mobility shift assay using mutated oligos and supershift assays with Sp1 and Sp3 antibodies demonstrated that both proteins specifically bound to this promoter region. Moreover, we found that CENP-W was highly induced by serum stimulation followed by serum deprivation, with Sp1 and Sp3 transcription factors involved in this transactivation. Taken together, our results suggest that Sp1 together with Sp3 may function as the main regulator of the basal and serum-induced transcription of CENP-W.