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 myofibril-binding 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.

Figure 1

Summary diagram giving a schematic representation of the domain organizations for M-protein and for myosin. The presentation emphasizes the modular construction of M-protein from repetitive Ig cII (cross-hatched rectangles) and fibronectin type III repeats (shaded rectangles) interspersed by unique sequence stretches of varying length. Recombinant constructs used for mapping of binding sites, transfection of cultured myoblasts, and phosphorylation assays are marked by brackets. The location of the most prominent proteolytic fragments of myosin is given in the lower panel above the myosin sketch. Brackets indicate the borders of the recombinant LMM constructs used in this study.

Figure 2

Binding of M-protein and its proteolytic 45-kDa fragment to myosin rod. (A) SDS-PAGE analysis (4–14%) of M-protein purified from bovine skeletal muscle (lane 1) and gel filtration fractions of an endoproteinase Asp-N digest of M-protein (see MATERIALS AND METHODS) (lanes 2–12). M = molecular mass standards in kilodaltons. (B) M-protein (1) and corresponding fractions of the M-protein digest (2–12) spotted on nitrocellulose filters after incubation with biotinylated myosin-rod or LMM 30. Note that only native M-protein (1) and its 45-kDa fragment (comprising domains Mp2–Mp4; fractions 9–11) show binding to myosin rod, while the proteolytic 110-kDa fragment of M-protein (comprising domains Mp5–Mp13; lanes 2–6) does not bind to myosin rod (the positions of these proteolytic fragments are indicated by arrows). Note also that both M-protein fragments do not bind to LMM30. Thus myosin rod binding of M-protein requires domains Mp2–Mp4.

Figure 3

Binding of recombinant M-protein fragments to proteolytic and recombinant derivatives of myosin. (A) SDS-PAGE analysis (6–20% gradient gels) of purified recombinant M-protein fragments: Mp2 (lane 1), Mp2–Mp3 (lane 2), Mp3 (lane 3), and Mp4 (lane 4). M = molecular mass standards in kilodaltons. For domain structure of M-protein see Figure 1. (B) Results of binding assays. The same amounts (ca. 1 μg) of myosin rod, proteolytic LMM, LMM 75, LMM 59, LMM 50, LMM 30, LMM 50–75, and ovalbumin, serving as a control, were spotted on nitrocellulose filters and overlaid with increasing concentrations (1 = 0.5 μM, 2 = 1.5 μM, 3 = 4.5 μM) of M-protein fragments Mp2 (a), Mp2 to Mp3 (b), Mp3 (c), and Mp4 (d). (e) Control without protein in the overlay buffer. Binding of M-protein fragments carrying the carboxy-terminal EEF-tag was detected with monoclonal antibody YL1/2, which specifically recognizes this tag. Note the specific binding of M-protein fragment Mp2–Mp3 to all myosin fragments that contain the central portion of LMM (myosin heavy chain residues 1506–1674) but not to LMM30 and LMM50–75.

Figure 4

Phosphorylation of native M-protein and phosphoamino acid analysis of M-protein and a recombinant Mp1–5 fragment. (A) M-protein from bovine skeletal muscle (lane 1) and a limited digest with trypsin (lane 2), analyzed on 4–12% SDS-PAGE (see MATERIALS AND METHODS). (B) The corresponding autoradiograph of the samples shown in panel A after incubation with PKA in the presence of [γ-³²P] ATP shows that M-protein (lane 1) is readily phosphorylated while its tryptic fragment (Mp6– Mp13) is not (lane 2). M = molecular mass standards in kilodaltons. (C) Phosphoamino acid analysis of native M-protein from bovine skeletal muscle (lane 1) and the recombinant M-protein fragment Mp1–Mp5 (lane 2) after phosphorylation with PKA. Positions of marker amino acids are indicated. Clearly, phosphorylation occurs in both samples exclusively on serine residues.

Figure 5

In vitro phosphorylation of mutant recombinant M-protein fragments. Purified EEF-tagged Mp1–Mp5 fragments were phosphorylated in vitro by PKA and ³²P-labeled ATP and run on two identical 4–10% gradient polyacrylamide gels. One gel was blotted to nitrocellulose and stained with the antibody recognizing the EEF-tag and a peroxidase-coupled secondary antibody (lanes 1–4), while the second gel was dried and autoradiographed (lanes 5–8). Lanes 1–4 contain approximately identical amounts of Mp1–Mp5 (lane 1), Mp1–Mp5 (Ser39/Ala) (lane 2), Mp1–Mp5 (Ser76/Ala) (lane 3), and Mp1–Mp5 (Ser39, 76/Ala) (lane 4). Lanes 5–8 show that Mp1–Mp5 and its (Ser39/Ala) mutant are phosphorylated, while phosphorylation of both mutants containing the Ser76/Ala mutation (lanes 7 and 8) is almost completely abolished. Thus Ser76 is the PKA phosphorylation site of M-protein.

Figure 6

Interaction of unphosphorylated and PKA phosphorylated M-protein with proteolytic and recombinant derivatives of myosin. The same amounts (1 μg) of myosin-rod, proteolytic LMM, LMM75, LMM59, LMM50, LMM30, LMM50–75, and ovalbumin, serving as a control, were spotted on nitrocellulose filters and overlayed with increasing concentrations (1 = 0.1 μM, 2 = 0.3 μM, 3 = 1.0 μM) of unphosphorylated (a) and phosphorylated (b) M-protein. (c) is a control without protein. Binding of M-protein was detected using the monoclonal M-protein antibody MpAA280 (see Obermann, et al., 1996). Note that phosphorylation of M-protein almost completely abolished binding to myosin derivatives.

Figure 7

Expression of recombinant M-protein fragments in transiently transfected BHK-21/C13 cells. BHK-21/C13 cells transfected with constructs encoding Mp2 (A and B), Mp3 (C and D), or Mp2–Mp3 (E–H) were double stained with T7-tag antibody to localize the recombinant M-protein fragment (A, C, E, and G) and a titin antibody (B, D, F, and H). (A– D) Examples of transfected cells that contain large amounts of diffusely distributed recombinant protein (A and C) and titin, which appears in a normal staining pattern (arrowheads in B and D). The expressed Mp2–Mp3 construct either disrupts MLS, which then results in the appearance of numerous cytoplasmic aggregates containing both the recombinant protein and titin (arrowheads in E and F), or colocalizes with MLS (arrowheads in G and H). For details see RESULTS. Magnification, 1050×.

Figure 8

Expression of recombinant M-protein fragments in differentiating C2C12 cells. C2C12 cells transfected with constructs encoding T7-tagged Mp2–Mp3 (A–D) or Mp9–Mp13 (E) were allowed to differentiate for 2 (E) or 6 (A to D) d, respectively. Subsequently they were stained with T7-tag antibody (A, C, and E) and tetramethylrhodamine-5-isothiocyanate-labeled phalloidin (B) or MpAA259 (D) followed by secondary antibody. Note that most of the expressed Mp2–Mp3 polypeptide associates with myofibrils (arrowheads in A–D). Transfection with Mp9–Mp13 leads to expression of a polypeptide that binds to myofibrils in a periodic manner (arrowheads in E). For details see RESULTS. Magnification, 1050×.