Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor. dystroglycan (DG) The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal acrin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polanty-regulating kinase, PAR-1b. interacts with the utrophin-DG complex, and positively regulates the interaction between Utrophin and DG In this study. we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with clystrophin at the skeletal muscle membrane These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain (C) 2009 Elsevier Inc. All rights reserved.