The muscle regulatory and structural protein MLP is a cytoskeletal bindingpartner of beta I-spectrin

Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural int egrity of the contractile apparatus. The ability of MLP to regulate myogene sis is particularly interesting since it exhibits multiple subcellular loca lizations, being found ill both nuclear and cytoplasmic compartments. Despi te extensive biochemical analyses on MLP, the mechanism(s) by which it infl uences the myogenic program remains largely undefined. To further examine t he role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein beta 1-spectrin was isolated. Protein interaction assays demonstrate that MLP and beta 1-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. beta I-spectrin binds specifically to MLP but not to the MLP re lated proteins CRP1 and CRP2 or to other LIM domain containing proteins. Th e MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin. Confocal microscopy studies also reveal t hat MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that beta-spectrin is a known costamere protein, we propose that sarcolemm a-associated MLP also serves as a key costamere protein, stabilizing the as sociation of the contractile apparatus with the sarcolemma by linking the b eta-spectrin network to the alpha-actinin crosslinked actin filaments of th e myofibril.