EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40

Dp40 is the shortest DMD gene product that has been reported to date. It is encoded by exons 63-70, a region required for a beta-dystroglycan interaction. Its expression has been identified in rat, mouse, and human; however, its function remains unknown. To explore the expression of Dp40 transcript and subcellular localization of epitope-tagged Dp40 proteins, RT-PCR and immunofiuorescence assays were performed in PC12 cells. The expression of Dp40 mRNA was found in undifferentiated and nerve growth factor-differentiated PC12 cells. According to immunofluorescence analyses, the recombinant protein Dp40 was mainly localized in the cell periphery/cytoplasm of undifferentiated and differentiated PC12 cells, a small amount of this protein is localized to the nucleus of differentiated cells. With the aim to identify the amino acids involved in the nuclear localization of Dp40, an in silico analysis was performed and it predicted that prolines 93 and 170, located within EF1 and EF2-hand domains, are involved in the nuclear localization of this protein. This prediction was confirmed by site-directed mutagenesis, the Dp40-L93P mutant was localized to the nucleus and cell periphery, while Dp40-L170P and Dp40-L93/170P showed mainly a nuclear localization. Dp40 co-localizes with beta-dystroglycan and the co-localization score was statistically reduced in Dp40-L93P, Dp40-L170P and Dp40-L93/170P mutants. (C) 2015 Elsevier Ireland Ltd. All rights reserved.