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Kinase-related protein (telokin) is phosphorylated by smooth-muscle myosin light-chain kinase and modulates the kinase activity.

ABSTRACT: 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.

SUBMITTER: Sobieszek A

PROVIDER: S-EPMC1218937 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Project description:Telokin, an abundant gizzard protein, inhibited phosphorylation of regulatory light chain when filamentous myosin was used as the substrate but no inhibition was observed with myosin subfragment 1. At physiological telokin-to-myosin molar ratio (1:1), the inhibition amounted to a 3.5-fold reduction in the initial phosphorylation rate whereas at high molar excess of telokin over myosin, we observed an up to 20-fold decrease in this rate. In agreement with previous observations [Shirinsky, Vorotnikow, Birukov, Nanaev, Collinge, Lukas, Sellers and Watterson (1993) J. Biol. Chem. 268, 16578-16583], telokin did not inhibit phosphorylation of the isolated regulatory light chain of myosin and only moderately (35%) inhibited that of heavy meromyosin. To gain a better understanding of the mechanism of this inhibition, we investigated the effects of telokin on the recently described [Babiychuk, Babiychuk and Sobieszek (1995) Biochemistry 34, 6366-6372] oligomeric properties of smooth-muscle myosin light-chain kinase (MLCK). We showed, on the one hand, that telokin rapidly solubilized the large kinase oligomers formed at low ionic strength. With soluble kinase, on the other hand, telokin acted to increase the relative concentration of MLCK dimers and to decrease that of the hexamers and octamers. This, in turn, resulted in a reduction in the amount of MLCK bound to myosin because filamentous myosin appeared to exhibit a higher affinity for the hexamers than for the dimers. Telokin by itself was also shown to dimerize and oligomerize in solution and this oligomerization was greatly enhanced in the presence of MLCK. We suggest that telokin affects myosin phosphorylation by modulation of the oligomeric state of MLCK and its interaction with myosin filaments.

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