PHOSPHORYLATION OF MYOSIN REGULATORY LIGHT-CHAINS BY THE MOLLUSCAN TWITCHIN KINASE

The unusually large (approximate to 600 to >3000 kDa) myosin-associate d proteins of the titin/twitchin superfamily are considered to be impo rtant cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60-240 modular fibronectin-typ e-III and immunoglobulin-C2 repeats in these proteins which further co ntain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitch in was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19-kDa myosin regulato ry light chains. The twitchin kinase phosphorylated purified light cha ins on Thr15 in a region which shared a high degree of similarity with the phosphorylation site fur vertebrate smooth muscle myosin light ch ain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the fi rst potential substrate for any of the giant protein kinases and suppo rt a dual role of twitchin in molluscan muscle as a cytoskeletal prote in as well as a myosin light chain kinase.