Obscurin Is a Ligand for Small Ankyrin 1 in Skeletal Muscle

Abstract

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

“…Of special interest is the fact that obscurin is present on the lateral surfaces of nascent myofibrils and between the forming narrow myofibril bundles. Yeast two-hybrid assay screening recently showed that obscurin interacts with small ankyrin 1, and immunolabeling revealed close localization of these two proteins in skeletal muscle fibers (Bagnato et al 2003;Kontrogianni-Konstantopoulos et al 2003;Armani et al 2006). Small ankyrin 1 is a ligand protein structurally associated with the sarcoplasmic reticulum (Porter et al 2005).…”

“…In recent years, this antibody was used in a large number of laboratories for detection of sarcomeric α-actinin in cardiac and skeletal muscle in vivo and in cell culture (e.g. Bagnato et al 2003;Sanger et al 2005;Musa et al 2006;Kontrogianni-Konstantopoulos et al 2003.…”

“…The complex primary structure of the gene encoding obscurin suggests that it has multiple functions (Bang et al 2001;Young et al 2001;Russell et al 2002;Fukuzawa et al 2005). Obscurin interacts with titin (Bang et al 2001, Young et al 2001) and small ankyrin 1 (Bagnato et al 2003;Kontrogianni-Konstantopoulos et al 2003). Earlier we found that the expression of obscurin gene is rapidly up-regulated during progressive heart hypertrophy (Borisov et al 2003).…”

Isoform Specificity among Ankyrins

“…The ␣-helix surface (residues 1773, 1777, 1780, 1784, and 1788) may also associate with signaling proteins such as GTPases or protein kinases, which may not be easily detected in conventional yeast two-hybrid assays (28,29). The giant Rho-guanine exchage factor obscurin has been reported to bind to a similar but not identical site in small ankyrin-1 (30,31). Therefore, the ankyrin-B amphipathic ␣-helix and surrounding residues may accommodate multiple protein interactions to regulate ankyrin-B function.…”

The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere

“…Obscurin is the third member of the family of sarcomeric giants, along with titin and nebulin, and contains Ig-Fn modules and multiple signaling domains [51,52]. While its localization in the sarcomere remains controversial [51], the interactions with titin [51], myosin [49] and ankyrin [53] suggest that, similar to its invertebrate ortholog Unc-89 [52], obscurin might provide an elastic connection between the M-band and the plasma membrane and/or between the M-bands of adjacent myofibrils.…”