Titin-cap associates with, and regulates secretion of, Myostatin.
Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth. To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins. Using a yeast two-hybrid screen, we identified Titin-cap (T-cap) protein as interacting with Myostatin. T-cap is a sarcomeric protein that binds to the N-terminal domain of Titin and is a substrate of the titin kinase. Mammalian two-hybrid studies, in vitro binding assays and protein truncations in the yeast two-hybrid system verified the specific interaction between processed mature Myostatin and full-length T-cap. Analysis of protein-protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T-cap with a KD of 40 nM. When T-cap was stably overexpressed in C(2)C(12) myoblasts, the rate of cell proliferation was significantly increased. Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T-cap, but an increase in the retention of mature Myostatin indicated that T-cap may block Myostatin secretion. Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T-cap contained lower levels of Myostatin. Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T-cap could thus be due to reduced Myostatin secretion. These results suggest that T-cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin.
Nicholas
Gina
G
Animal Genomics, AgResearch, East Street, Hamilton, New Zealand.
Thomas
Mark
M
Langley
Brett
B
Somers
Wayne
W
Patel
Ketan
K
Kemp
C Fred
CF
Sharma
Mridula
M
Kambadur
Ravi
R
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D064211:Connectin; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D017930:Genes, Reporter; D007700:Kinetics; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D055435:Myostatin; D011485:Protein Binding / Q000502:physiology; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D020349:Surface Plasmon Resonance; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D064211:Connectin; D017077:Culture Media, Conditioned; C525912:Mstn protein, mouse; D009124:Muscle Proteins; D055435:Myostatin; C577294:Tcap protein, mouse; D016212:Transforming Growth Factor beta