Four and a half LIM protein 1 binds myosin-binding protein C and regulates myosin filament formation and sarcomere assembly.
Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced "sac-like" myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.
McGrath
Meagan J
MJ
Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
Cottle
Denny L
DL
Nguyen
Mai-Anh
MA
Dyson
Jennifer M
JM
Coghill
Imogen D
ID
Robinson
Paul A
PA
Holdsworth
Melissa
M
Cowling
Belinda S
BS
Hardeman
Edna C
EC
Mitchell
Christina A
CA
Brown
Susan
S
D000205:Actomyosin / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D009218:Myosins / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D012518:Sarcomeres / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000099163:Myosin Binding Protein C
D000205:Actomyosin; D002352:Carrier Proteins; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D009218:Myosins; D009841:Oligonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D000099163:Myosin Binding Protein C; C101149:FHL1 protein, human