Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Biochemistry. 1994 Jan 25;33(3):840-7.

Mutagenesis of the phosphorylation site (serine 19) of smooth muscle myosin regulatory light chain and its effects on the properties of myosin.

Author information

  • 1Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.

Abstract

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)

PMID:
8292613
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Write to the Help Desk