Autophosphorylation of molluscan twitchin and interaction of its kinase domain with calcium/calmodulin.
An approximately 750-kDa member of the family of giant titin/twitchin-like myosin-associated proteins was highly purified from muscle of the marine mollusc Aplysia californica. Purified twitchin was able to autophosphorylate on threonine, which demonstrates its protein serine/threonine kinase activity. cDNA sequence analysis of the cloned kinase domain of molluscan twitchin revealed that it is most closely related with the kinase domains of Caenorhabditis elegans twitchin (62% identity) and vertebrate myosin light chain kinases (45% average identity). Analysis of the cDNA sequence further suggested the presence of a potential calmodulin-binding site in a putative autoinhibitory region. The functional activity of this site was demonstrated by the calcium-dependent binding of purified twitchin to immobilized calmodulin and the fact that this interaction could be competed with synthetic peptides deduced from the cDNA sequence. Furthermore, biotinylated calmodulin bound to immobilized twitchin in gel-overlay assays with nanomolar affinity (EC50 approximately equal to 70 nM). The potential regulation of twitchin by calcium/calmodulin indicates that titin-like molecules may serve dynamic functions during contraction-relaxation cycles in muscle in addition to their functions as cytoskeletal proteins.
Heierhorst
J
J
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029.
Probst
W C
WC
Vilim
F S
FS
Buku
A
A
Weiss
K R
KR
D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D015801:Helminth Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D009126:Muscle Relaxation; D009132:Muscles / Q000378:metabolism / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D029742:Caenorhabditis elegans Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D015801:Helminth Proteins; D009124:Muscle Proteins; C489233:unc-22 protein, C elegans; D017346:Protein Serine-Threonine Kinases; D009219:Myosin-Light-Chain Kinase; D002118:Calcium