Mutagenesis of the phosphorylation site (serine 19) of smooth muscle myosin regulatory light chain and its effects on the properties of myosin.
A full-length cDNA of smooth muscle regulatory light chain was obtained and the recombinant regulatory light chain was expressed in an Escherichia coli expression system. The recombinant regulatory light chain was introduced into myosin or HMM using a subunit exchange strategy [Morita, J., Takashi, R., & Ikebe, M. (1991) Biochemistry 30, 9539-9545]. The recombinant wild-type regulatory light chain exhibited the same biological properties as the natural isolate, i.e., phosphorylation at Ser-19 by myosin light-chain kinase and phosphorylation-activated actomyosin ATPase activity. To clarify whether or not the activation of the ATPase by phosphorylation is simply due to the introduction of negative charge, we produced three mutant light chains. Two of them contain Ser-19 substituted by either Asp or Ala and the third contains Asp substituted for both Thr-18 and Ser-19. Incorporation of the Asp mutant partially activated actomyosin ATPase activity but the activation level was significantly lower than that by phosphorylation. The Asp/Asp mutant further activated actomyosin ATPase activity. On the other hand, the Ala mutant did not affect the ATPase activity. Incorporation of Asp mutant slightly affected the 10S-6S conformational transition and filament formation of myosin. The Asp/Asp mutant more significantly affected the 10S-6S conformational transition and filament formation of myosin. These results suggested that the activation of smooth muscle myosin requires the introduction of negative charge in the defined spacial position. Using Ser-19 deficient mutants, the effects of Thr-18 phosphorylation on myosin function was also studied. Actin-activated ATPase activity of myosin was significantly activated by phosphorylation of Thr-18.(ABSTRACT TRUNCATED AT 250 WORDS)
Kamisoyama
H
H
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Araki
Y
Y
Ikebe
M
M
D000199:Actins / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D012694:Serine / Q000235:genetics
D000199:Actins; D011994:Recombinant Proteins; D012694:Serine; D011189:Potassium Chloride; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D009218:Myosins