Phosphorylation of dystrophin Dp71d by Ca2+/calmodulin-dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells.
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 mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation 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-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/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.
Calderilla-Barbosa
Luis
L
Genetics and Molecular Biology Department, Centro de Investigación y de Estudios Avanzados del IPN, San Pedro Zacatenco, Mexico.
Ortega
Arturo
A
Cisneros
Bulmaro
B
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats
D016189:Dystrophin; D019913:Nuclear Localization Signals; C085339:apo-dystrophin 1; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases