Ca++-Calmodulin-Dependent Phosphorylation of Myosin, and Its Role in Brush Border Contraction in vitro

• Published in: The Journal of cell biology 1982-12, Vol.95 (3), p.943-959
• Main Authors: Thomas C. S. Keller, III, Mooseker, Mark S.
• Format: Article
• Language: English
• Subjects: Actins; Adenosine Triphosphate - pharmacology; Adherens junctions; Animals; Calcium - pharmacology; Calcium-Binding Proteins - pharmacology; Calmodulin - pharmacology; Cell Membrane - physiology; Chickens; Epithelial cells; Epithelium - ultrastructure; Gels; Kinetics; Lexical contractions; Microfilaments; Microvilli; Microvilli - drug effects; Microvilli - physiology; Microvilli - ultrastructure; Movement; Myosin-Light-Chain Kinase; Myosins - metabolism; Phosphorylation; Physiological regulation; Protein Kinases - metabolism; Smooth muscle; Trifluoperazine - pharmacology
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.95.3.943

We have reinvestigated the effects of Ca++and ATP on brush borders isolated from intestinal epithelial cells. At 37°C, Ca++(1 μM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37°C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca++(1 μM), and occurs in both membrane-intact and demembranated brush borders. Ca++-dependent-solation of microvillus cores requires a concentration of Ca++slightly greater (10 μM) than that required for contraction. Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin ( BBMLC20), is stimulated by Ca++. At 37°C, BBMLC20phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC20phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca++regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca++- and calmodulin-dependent brush border myosin light chain kinase.