Phosphorylation of dystrophin Dp71d by Ca 2+ /calmodulin- dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells Luis Calderilla-Barbosa, Arturo Ortega and Bulmaro Cisneros Genetics and Molecular Biology Department, Centro de Investigacio ´n y de Estudios Avanzados del IPN, San Pedro Zacatenco, Mexico Abstract We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mecha- nisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of pro- teins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphoryl- ated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phos- phorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphory- lation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphtha- lenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca 2+ /diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca 2+ / calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization. Keywords: CaMKII, Dp71d, dystrophin, nuclear localization, PC12 cells, phosphorylation. J. Neurochem. (2006) 98, 713–722. Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene that result in either the absence or the dysfunction of dystrophin, a 427-kDa membrane-associated protein (Koenig et al. 1988; Ahn and Kunkel 1993). Dystrophin consists of four domains, an N-terminal actin-binding domain, a central spectrin-like rod domain, a cystein-rich region and a C-terminal domain (Davison and Critchley 1988; Hoffman et al. 1988; Blake et al. 2002). The cystein-rich and C-terminal domains associate with a group of transmembrane glycoproteins and cytoplasmic proteins known collectively as the dystrophin-associated protein complex (DAPC) (Yoshida and Ozawa 1990; Ervasti and Campbell 1991; Suzuki et al. 1992; Blake and Kroger 2000; Tommasi di Vignano et al. 2000). It is thought that dystrophin and the DAPC help to maintain the sarcolemmal stability and participate in the signaling transduction occurring from the Received February 3, 2006; revised manuscript received February 24, 2006; accepted February 27, 2006. Address correspondence and reprint requests to Bulmaro Cisneros PhD, Genetics and Molecular Biology Department, Centro de Investi- gacio ´n y de Estudios Avanzados del IPN, Avenue IPN #2508, San Pedro Zacatenco, 07360, Me ´xico. E-mail: bcisnero@cinvestav.mx Abbreviations used: Bis I, bisindolylmaleimide I; BSA, bovine serum albumin; C-20, anti-dystrophin polyclonal antibody dystrophin; CaM- KII, Ca 2+ /calmodulin-dependent protein kinase II; CK-II, casein kin- ase II; DAPC, dystrophin-associated protein complex; DMD, Duchenne muscular dystrophy; Dp71d, splicing isoform of Dp71; DTT, dithio- threitol; N/C ratio, nuclear/cytoplasmic ratio; NGF, nerve growth factor; PBS, phosphate-buffered saline; PKA, cAMP-dependent protein kinase; PKC, Ca 2+ /diacylglicerol-dependent protein kinase; PMSF, phenyl- methylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TBS-T, Tris- buffered saline with Tween; TPA, 2-O-tetradecanoyl phorbol 13-acetate; W-7, N-(6-aminohexyl)-1-naphthalenesulfonamide. Journal of Neurochemistry , 2006, 98, 713–722 doi:10.1111/j.1471-4159.2006.03904.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 98, 713–722 713