Myosin, actin and associated cytoskeletal proteins interact to form and maintain the large brush border membrane surface necessary for nutrients absorption in intestinal epithelial cells. In addition, actomyosin-based movements of microvilli and contractions of the terminal web region of the brush border may be important in the absorptive process itself. To define and characterize the molecular basis of these events, the role of chicken brush border myosin phosphorylation and of calcium binding to myosin will be studied in regulation of: a) myosin actin-activated ATPase activity, b) myosin filament assembly, c) the conformation of the myosin molecule, and brush border myosin-directed movement of latex beads in an in vitro motility assay. A brush border calmodulin-dependent heavy chain kinase, calmoudulin-dependent light chain kinase and calmoudulin- independent light chain kinase that we have recently identified and isolated will be used in these studies to phosphorylate purified brush myosin at specific, well-defined sites. The regulation of the activity of the calmodulin-dependent myosin kinases by calcium and calmodulin and of the heavy chain kinase by autophosphorylation will also be characterized. The heavy chain kinase which is the only heavy chain kinase known to be regulated by calmodulin, will be assayed for in extracts of other tissues and compared immunologically to the apparently similar and widely distributed calmoulin-dependent protein kinase II. Our previous characterization of the myosin phosphorylation sites by phosphoamino acid analysis and peptide mapping will be extended to the determination of the sequence around each site. In addition, the heavy chain phosphorylation site will be localized within the heavy chain. The regulation of contraction of isolated brush borders by myosin phosphorylation and by calcium will also be characterized by identification of the phosphorylation sites, by reconstitution of nyosin-depleted brush borders with phosphorylated myosin, and by use of specific antibody and peptide inhibitors of each kinase. In vivo phosphorylation sites will also be identified in chicken intestinal explants. These studies should enhance our understanding of myosin function and organization and their regulation in intestinal epithelial and other non-muscle cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032567-05
Application #
3281537
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1983-07-01
Project End
1989-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Swanljung-Collins, H; Collins, J H (1994) Brush border myosin I has a calmodulin/phosphatidylserine switch and tail actin-binding. Adv Exp Med Biol 358:205-13
Swanljung-Collins, H; Collins, J H (1992) Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation. J Biol Chem 267:3445-54
Swanljung-Collins, H; Collins, J H (1991) Rapid, high-yield purification of intestinal brush border myosin I. Methods Enzymol 196:3-11
Swanljung-Collins, H; Collins, J H (1991) Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound. J Biol Chem 266:1312-9
Borysenko, C; Rieker, J P; Swanljung-Collins, H et al. (1988) Phosphorylation of brush border myosin at threonine on its 20 kDa light chains by a calmodulin-independent kinase activates its ATPase. FEBS Lett 235:149-52
Rieker, J P; Swanljung-Collins, H; Collins, J H (1987) Purification and characterization of a calmodulin-dependent myosin heavy chain kinase from intestinal brush border. J Biol Chem 262:15262-8
Swanljung-Collins, H; Montibeller, J; Collins, J H (1987) Purification and characterization of the 110-kDa actin- and calmodulin-binding protein from intestinal brush border: a myosin-like ATPase. Methods Enzymol 139:137-48
Rieker, J P; Collins, J H (1987) Phosphorylation of brush border myosin by brush border calmodulin-dependent myosin heavy and light chain kinases. FEBS Lett 223:262-6
Rieker, J P; Swanljung-Collins, H; Montibeller, J et al. (1987) Isolation and characterization of calmodulin-dependent myosin heavy chain kinase from intestinal brush border. Methods Enzymol 139:105-14
Rieker, J P; Swanljung-Collins, H; Montibeller, J et al. (1987) Brush border myosin heavy chain phosphorylation is regulated by calcium and calmodulin. FEBS Lett 212:154-8