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Pull-down of Calmodulin-binding Proteins
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Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin

H Brzeska1, D Kulesza-Lipka, E D Korn

  • 1Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.

The Journal of Biological Chemistry
|November 25, 1992
PubMed

Insights

Calcium-calmodulin inhibits Acanthamoeba myosin I heavy chain kinase activity by blocking its binding to phospholipids. This regulation mechanism controls myosin I-dependent cell motility.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I is regulated by phosphorylation of their heavy chains.
  • Myosin I heavy chain kinase (MIHCK) activity is significantly enhanced by autophosphorylation and acidic phospholipids.

Purpose of the Study:

  • To investigate the regulatory role of Ca(2+)-calmodulin on MIHCK activity and binding.
  • To elucidate the mechanism by which Ca(2+) regulates myosin I-dependent motile activities.

Main Methods:

  • Biochemical assays to measure kinase activity.
  • Phospholipid binding assays using vesicles and plasma membranes.
  • Proteolytic digestion to identify binding domains.

Main Results:

  • Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of MIHCK.
  • Ca(2+)-calmodulin inhibits the catalytic activity of unphosphorylated MIHCK in the presence of phospholipids.
  • Micromolar Ca(2+)-calmodulin inhibits MIHCK binding to phospholipid vesicles and plasma membranes.
  • Removal of a 7-kDa NH2-terminal segment abolishes both calmodulin and phospholipid binding.

Conclusions:

  • Ca(2+)-calmodulin and phospholipids bind to the same or overlapping sites on the NH2-terminus of MIHCK.
  • Ca(2+) acts as an inhibitor of MIHCK activity and binding, providing a mechanism to regulate myosin I-dependent motility in vivo.

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