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, 16578Ő16583], 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, 6366Ő6372] 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.
Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain
