Myosin-associated giant protein kinases of the titinkwitchin-like superfamily have previously been implicated in the regulation of muscle function, based on genetic and physiological studies. We find that recombinant constitutively active Caenorhabditis eleguris and Aplysiu twitchin kinase fragments differ in their catalytic activities and peptide-substrate specificities, as well as in their sensitivities to the naphthalene sulfonamide inhibitors 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-l,4-diazepine (ML-7) and 1-(5-iodonaphthalenesulfonyl)-1H-hexahydro-l,4-diazepine (ML-9). The constitutively active Aplysia twitchin kinase fragment has a remarkably high activity (V,,,,, > 100 pmol . min-' . mg-') towards some substrate peptides. The autoinhibited forms of these twitchin kinases can be activated in a Ca"-dependent manner by the dimeric form of the SlOOAl protein (S100A1,). The twitchin kinase SIOOAI,-binding site can also bind Ca' +/calmodulin but neither kinase is activated by calmodulin. The data provide a functional basis for the ongoing crystallographic study of twitchin kinase fragments.Keywords: protein kinase; twitchin; titin ; S100; calmodulin.The myosin-associated giant proteins such as titin, twitchin and projectin are predominantly composed of repeating modular fibronectin-type-Ill-like and immunoglobulin-like domains [I -31. These domains are considered to be important for the cytoskeletal functions of these proteins as possible molecular rulers for the length and elasticity of the sarcomere 141. Titinltwitchinlike proteins also contain a single protein kinase domain [1-31, and mutant phenotypes of Caenorhubditis elegans twitchin [ 11 and Drosophila projectin [3] indicate that the kinase may be involved in the regulation of myosin activity.The twitchin kinase domain is distantly related to myosin light chain kinases (MLCKs) [ 11. Constitutively active recombinant twitchin kinases phosphorylate a MLCK substrate peptide [5, 61, and Aplysia regulatory myosin regulatory light chains (MLCs) have been identified as the first endogenous substrates for any of the giant protein kinases [6]. C. e1egun.s and Aplysia twitchin kinases contain an autoinhibitory region just C-terminal to the catalytic core 1.5581 similar to that of calmodulin-regulated kinases. Crystal structures of these kinases demonstrate that this region inhibits the kinase by an intrasteric mechanism

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