ASSOCIATION OF ACICULIN WITH DYSTROPHIN AND UTROPHIN

Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin , A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D ., and Koteliansky, V.E. (1994) J. Cell Sci. 107, 159-173). Aciculin e xpression in skeletal muscle is developmentally regulated, and this pr otein is particularly enriched at cell-matrix adherens junctions of mu scle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1 993-2003). The purpose of our study was to identify cytoskeletal prote in(s) interacting with aciculin in various cell types. Using immunopre cipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) similar to 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigr ated with dystrophin and immunoblotted with anti-dystrophin antibodies . The association between aciculin and dystrophin in C2C12 cells was s hown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the r everse immunoprecipitation experiments, aciculin was detected in the p recipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes show ed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and ma ture C2C12 myotubes with antibodies against aciculin and dystrophin re vealed precise colocalization of these two cytoskeletal proteins throu ghout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myote ndinous junctions. In contrast, utrophin, an autosomal homologue of dy strophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these prot eins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aci culin-associated protein in skeletal muscle, immunoblotting of anti-ac iculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle c ells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculi n is a major utrophin-binding protein in this cell type. Taken togethe r, our data show that aciculin is a novel dystrophin- and utrophin-bin ding protein. Association of aciculin with dystrophin (utrophin) in va rious cell types might provide an additional cytoskeletal-matrix trans membrane link at sites where actin filaments terminate at the plasma m embrane.