Modulation of myosin filament activation by telokin in smooth muscle liberation of myosin kinase and phosphatase from supramolecular complexes

• Published in: Biophysical chemistry Vol. 113; no. 1; pp. 25 - 40
• Main Authors: Sobieszek, Apolinary, Andruchov, Oleg Y, Grabarek, Zenon, Kulikova, Natalia, Liebetrau, Claudia, Matusovsky, Oleg S
• Format: Journal Article
• Language: English
• Published: Netherlands 01.01.2005
• Subjects: Actin Cytoskeleton - metabolism; Actomyosin - metabolism; Animals; Catalytic Domain; Macromolecular Substances - metabolism; Muscle, Smooth - metabolism; Myosin-Light-Chain Kinase - metabolism; Myosin-Light-Chain Phosphatase - metabolism; Myosins - antagonists & inhibitors; Myosins - chemistry; Myosins - metabolism; Peptide Fragments; Peptides - metabolism; Phosphorylation; Protein Binding; Substrate Specificity; Turkey
• ISSN: 0301-4622
• DOI: 10.1016/j.bpc.2004.07.038

The mechanism of telokin action on reversible phosphorylation of turkey gizzard myosin was investigated using a native-like filamentous myosin. This myosin contained endogenous calmodulin (CaM) and myosin light chain kinase (MLCK) at a molar ratio to myosin of about 1 to 40 or less depending on the initial extractions conditions. These levels were sufficient to fully phosphorylate myosin within 20-40 s or less after addition of [gamma-32P]ATP, but when the ATP was depleted, they became dephosphorylated indicating the presence of myosin light chain phosphatase (MLCP). Addition of telokin at the 1 to 1 or higher molar ratio to myosin caused a three- to five-fold inhibition of the initial phosphorylation rates (without reduction of the overall extent of phosphorylation) and produced a similar increase in the rate of dephosphorylation. The inhibition was also observed for myosin filaments free of MLCK and CaM together with constitutively active MLCKs produced by digestion, or by expression of a truncated mammalian kinase as well as for the wild-type enzyme. Thus, neither N- nor C-terminal of MLCK was necessary for interaction of myosin with telokin and the inhibition resulted from telokin-induced change of myosin head configuration within the filament that prevented their ordered, paracrystaline-like, aggregation. Sedimentation of the filamentous myosin in glycerol gradients showed that this change made the filaments less compact and facilitated release of the endogenous MLCK/CaM complex. For a mixture of the filaments with or without the complex, the configuration change resulted in an increase of the phosphorylation rate but not in its inhibition. The increase of the rate resulting from the liberation of the complex was also observed in mixtures of the filamentous myosin with added isolated regulatory light chain (ReLC) or soluble myosin head subfragment. This observation reinforces the above conclusions. The acceleration of the MLCP activity by telokin was shown to result from dissociation of its catalytic subunit from a MLCK/MLCP complex bound to the filamentous myosin. Analogous desensitizing effects of telokin were also demonstrated for the contraction and relaxation cycle of Triton-skinned fibers from guinea pig Teania coli. Taken together, our results indicate that telokin acted as an effective modulator or chaperone of the myosin filament and a scheme for its action in smooth muscle was proposed.