Substrate Specificity and Inhibitor Sensitivity of Ca²⁺/S100‐dependent Twitchin Kinases

Abstract

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

Calcium transients regulate patterned actin assembly during myofibrillogenesis

“…Alternatively, ML-7 may disrupt thin filament organization by means of inhibition of the titin kinase domain. This idea is supported by the observations that (1) ML-7 inhibits twitchin kinase, the invertebrate homologue of titin (Heierhorst et al, 1996b), (2) the high structural similarity between the MLCK and titin kinase domains (Mayans et al, 1998), and (3) the different doseresponse effects of ML-7 on thin versus thick filament assembly (this study). As mentioned previously, the titin kinase domain is initially located at the Z-disc during thin filament organization and may phosphorylate substrate(s) necessary for anchoring and/or nucleating F-actin polymerization.…”

Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase

“…Phosphorylation assays used 30 ng∕mL kinase samples and 0.2 mg∕mL of a peptide substrate (KKRARAATSNVFS) derived from kMLC11-23 (25). At time points, reaction mixture was withdrawn, spotted, and phosphoimaged.…”

Erratum: Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori

“…Tyrosine kinase assays were done with 100 g ml −1 kin1 and 1 l cell lysate supernatant in 20 mM MOPS, pH 7, 5 mM MnCl 2 , 1 mM Na 3 VO 4 , 0.1 mM ATP and 2 Ci [g-32 P]ATP, 3,000 Ci mM −1 , as described 33 . Dried gels were autoradiographed for 2-12 h. Quantification of calmodulin-and S100-induced activation and basal activity was done as described 26 . Phosphoproteins were enriched for microsequencing by chelating chromatography 27 and by SDS-PAGE.…”

“…1), excluding the possibility of a related activation mechanism. The most significant difference in the phosphorylation site on telethonin, as compared with the sites of phosphorylation of substrates for MLCK and twitchin 26 is the presence of an arginine in the P þ 1 position. A potential function of the phosphorylated Y170 could, therefore, be to directly bind this arginine, explaining the unusual presence of arginine in the P þ 1 position of the titin kinase substrate and the location of the titin kinase phosphorylation site, Y170, in the P þ 1 loop.…”

“…The addition of Ca 2+ /S100A1, Ca 2+ /CACY or Ca 2+ /CAPL has no activating effect; the presence of Ca 2+ /CACY suppresses basal titin-kinase activity by about tenfold, indicating that S100 proteins indeed modulate the activity of titin kinase. The addition of 10 M Zn 2+ in the assays using S100 proteins did not increase kinase stimulation but inhibited kin1(Y170E) as it inhibited C. elegans twitchin 26 .…”