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Twitchin from molluscan catch muscle: primary structure and relationship between site-specific phosphorylation and
Daisuke Funabara1, Shugo Watabe, Susan U Mooers
1Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
The Journal of Biological Chemistry
|May 21, 2003
Summary
Phosphorylation of twitchin, a protein in molluscan smooth muscle, is crucial for maintaining muscle force. Specific phosphorylation sites (D1 and D2) on twitchin are necessary for muscle relaxation from a sustained force state.
Area of Science:
- Muscle Physiology
- Molecular Biology
- Biochemistry
Background:
- Twitchin, a myosin thick filament-associated protein, plays a key role in regulating catch force in molluscan smooth muscle.
- The phosphorylation state of twitchin is a critical factor in the maintenance of this sustained muscle force.
Purpose of the Study:
- To obtain the full-length cDNA sequence of twitchin from the mussel Mytilus anterior byssus retractor muscle.
- To characterize the molecular structure and identify key functional motifs and phosphorylation sites within twitchin.
- To correlate twitchin's phosphorylation state with the mechanical properties of smooth muscle, specifically during relaxation from catch force.
Main Methods:
- Polymerase Chain Reaction (PCR) and 5' rapid amplification of cDNA ends (5' RACE) were used to obtain the full-length twitchin cDNA.
- Bioinformatic analysis was performed to determine the derived amino acid sequence and identify protein motifs.
- Specific antibodies against phosphorylation sites D1 and D2 were utilized to assess twitchin's phosphorylation state in correlation with muscle mechanical properties.
Main Results:
- The full-length twitchin cDNA sequence was obtained, revealing a large protein (approximately 530 kDa) with a complex domain arrangement including Ig, IgFn, and a Kinase domain.
- Key motifs were identified, including a Pro/Glu/Val/Lys-rich region and a DFRXXL motif potentially involved in myosin light chain kinase binding.
- Two major phosphorylation sites, D1 and D2, were localized in linker regions between specific immunoglobulin domains.
Conclusions:
- The phosphorylation of both D1 and D2 sites on twitchin is essential for the relaxation of molluscan smooth muscle from the catch state.
- Understanding twitchin phosphorylation provides insights into the molecular mechanisms underlying sustained muscle contraction and force maintenance.