Kinase-related protein (telokin) is phosphorylated by smooth-muscle myosin light-chain kinase and modulates the kinase activity.
Telokin is an abundant smooth-muscle protein with an amino acid sequence identical with that of the C-terminal region of smooth-muscle myosin light-chain kinase (MLCK), although it is expressed as a separate protein [Gallagher and Herring (1991) J. Biol. Chem. 266, 23945-23952]. Here we demonstrate that telokin is also similar to smooth-muscle myosin regulatory light chain (ReLC) not only in its gross physical properties but also as an MLCK substrate. Telokin was slowly phosphorylated by MLCK in the presence of Ca2+ and calmodulin and could be readily dephosphorylated by myosin light-chain phosphatase. A threonine residue was phosphorylated with up to 0.25 mol/mol stoichiometry. This low stoichiometry, together with the observed dimerization of telokin [Sobieszek and Nieznanski (1997) Biochem. J. 322, 65-71], indicates that the telokin dimer was acting as the substrate with a single protomer being phosphorylated. Our enzyme kinetic analysis of the phosphorylation reaction confirms this interpretation. Because telokin phosphorylation also required micromolar concentrations of MLCK, which also facilitates the formation of kinase oligomers, we concluded that the oligomers are interacting with telokin. Thus it seems that telokin modulates the phosphorylation rate of myosin filaments by a mechanism that includes the direct or indirect inhibition of the kinase active site by the telokin dimer, and that removal of the inhibition is controlled by slow phosphorylation of the telokin dimer, which results in MLCK dimerization.
Sobieszek
A
A
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
Andruchov
O Y
OY
Nieznanski
K
K
D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D008387:Marine Toxins; D052998:Microcystins; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments; D010455:Peptides; D010456:Peptides, Cyclic / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity
D002147:Calmodulin; D008387:Marine Toxins; D052998:Microcystins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010446:Peptide Fragments; D010455:Peptides; D010456:Peptides, Cyclic; C060640:telokin; D009219:Myosin-Light-Chain Kinase; C057862:cyanoginosin LR; D002118:Calcium