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.
