Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2.
During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.
Lange
Stephan
S
Institute of Cell Biology, ETH Hönggerberg, 8093 Zurich, Switzerland.
Auerbach
Daniel
D
McLoughlin
Patricia
P
Perriard
Evelyne
E
Schäfer
Beat W
BW
Perriard
Jean-Claude
JC
Ehler
Elisabeth
E
D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D064211:Connectin; D003402:Creatine Kinase / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D025221:Phosphofructokinases / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D064211:Connectin; C491381:Fhl2 protein, rat; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D014157:Transcription Factors; D011494:Protein Kinases; D025221:Phosphofructokinases; D003402:Creatine Kinase; D000263:Adenylate Kinase