AUTOPHOSPHORYLATION OF MOLLUSCAN TWITCHIN AND INTERACTION OF ITS KINASE DOMAIN WITH CALCIUM CALMODULIN/

An similar to 750-kDa member of the family of giant titin/ twitchin-li ke myosin-associated proteins was highly purified from muscle of the m arine mollusc Aplysia californica. Purified twitchin was able to autop hosphorylate on threonine, which demonstrates its protein serine/threo nine kinase activity. cDNA sequence analysis of the cloned kinase doma in 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). Anal ysis of the cDNA sequence further suggested the presence of a potentia l calmodulin-binding site in a putative autoinhibitory region. The fun ctional activity of this site was demonstrated by the calcium-dependen t binding of purified twitchin to immobilized calmodulin and the fact that this interaction could be competed with synthetic peptides deduce d from the cDNA sequence. Furthermore, biotinylated calmodulin bound t o immobilized twitchin in gel overlay assays with nanomolar affinity ( EC(50) congruent to 70 nM). The potential regulation of twitchin by ca lcium/calmodulin indicates that titin-like molecules may serve dynamic functions during contraction-relaxation cycles in muscle in addition to their functions as cytoskeletal proteins.