Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch.
Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini, respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.
Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan.
Daisuke
D
Funabara
Chieko
C
Hamamoto
Koji
K
Yamamoto
Akinori
A
Inoue
Miki
M
Ueda
Rika
R
Osawa
Satoshi
S
Kanoh
David J
DJ
Hartshorne
Suechika
S
Suzuki
Shugo
S
Watabe
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001696:Biomechanical Phenomena; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors; D002645:Chickens; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008974:Mollusca / Q000187:drug effects / Q000648:ultrastructure; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D009129:Muscle Tonus / Q000187:drug effects / Q000502:physiology*; D009132:Muscles / Q000187:drug effects / Q000648:ultrastructure; D050505:Mutant Proteins / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D009124:Muscle Proteins; D050505:Mutant Proteins; D010455:Peptides; D014335:Tropomyosin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins