Mapping of a Myosin-binding Domain and a Regulatory Phosphorylation Site in M-Protein, a Structural Protein of the Sarcomeric M Band

Abstract

The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2-Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibrilbinding site was uncovered in domains Mp9 -Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.

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

“…According to observations on isolated filaments, myomesin and M-protein bind to the central zone of myosin filaments [16] and to the C-terminus of titin [17], indicating participation in the sarcomeric cytoskeleton. The approximate positions of titin, myomesin and M-protein in the sarcomere were determined by biochemical assays and EM epitope localization; these data led to a molecular model of the M-band [11,18,19]. In this model, M-protein molecules bridge the myosin filaments at the level of the central M1 line (see Box 1), which is consistent with previous EM observations [20,21].…”

“…Similar to the PEVK fragment of titin, the EH-fragment is mostly unfolded and functions like an entropic spring [31] (Box 2). Phosphorylation sites (P, blue circles) [18,19] and interacting partners (identified above brackets) [11,18,30,69] are indicated.…”

Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeres

“…Within the A-band titin is tightly linked to the thick filament via its multiple binding sites for myosin-binding protein C (MyBP-C; Labeit et al, 1992;Houmeida et al, 1995;Freiburg and Gautel, 1996). The titin-myosin interaction is reinforced at the M-band where titin interacts with myomesin and M-protein-both relevant for the assembly and structural maintenance of thick filaments (Bähler et al, 1985;Nave et al, 1989;Vinkemeier et al, 1993;Obermann et al, 1996). Thus, titin's integration into the sarcomeric lattice is mediated by its interaction with multiple structural proteins along the half-sarcomere and provides an elastic connection between the thick and thin filament systems, thereby centering the A-band in the sarcomere (Houmeida et al, 1995).…”

“…According to the premyofibril model, the initial formation of regular sarcomeres involves the polymerization of actin, incorporation of myosin, as well as assembly and alignment of Z-bodies, which incorporate titin's N terminus and form the future Z-disc (Rhee et al, 1994;Sanger et al, 2000;Du et al, 2003). Subsequently titin's C terminus is integrated into the M-band and connected to the muscle myosin filament (Nave et al, 1989;Obermann et al, 1996). The resulting continuous filament system has been regarded as a molecular ruler and as a blueprint for sarcomere assembly because titin's PEVK-region, immunoglobulin, fibronectin, and kinase domains are associated with specific sections of the half-sarcomere and thus sublocalize the various titin-binding proteins along the myofilament (Labeit and Kolmerer, 1995;Trinick, 1996;van der Loop et al, 1996;Obermann et al, 1997;Gregorio et al, 1998).…”

Divergent polarization mechanisms during vertebrate epithelial development mediated by the Crumbs complex protein Nagie oko

“…Appropriate primers were used to amplify the cDNA encoding this portion of the protein and to clone the fragment into pET23/T7, a modified pET23a vector (Obermann et al, 1998). The His-tagged protein was expressed in E. coli BL21-CodonPlus(DE3)-RIL (Novagen), solubilized using a buffer containing 6 M guanidine-HCl and purified as described (Chumpia et al, 2003).…”