LLC-PK?1? is an epithelioid cell line that differentiates transport functions in postconfluent cells. A-system amino acid transport is very active in rapidly growing cells, but is stepped down to stable levels in postconfluent cells while Na?+?-dependent hexose transport develops to a high degree. The tumor promoter TPA rapidly reactivates the A-system. TPA also activates protein kinase C (C-kinase), a Ca?++?- and phosphatidylserine-dependent kinase activated by diacylglycerols (DG). TPA can substitute for DG. Certain DG analogs will also reactivate the A-system in vivo. In LLC-PK?1? cells, TPA treatment leads to redistribution of C-kinase from cytosol to the particulate (membranes and organelles) fraction. By fractionation experiments, the cellular loci of TPA-dependent redistribution will be defined more closely and compared to the effects of the DG analogs. Fluorescent and colloidal gold-labeled antibodies to purified C-kinase will be used in both light and electron microscopy, respectively, to examine C-kinase localization in untreated, TPA-treated, and lipid-treated postconfluent cells. Basolateral membranes (locus of the A-system) and/or endocytic vesicles will be isolated and the A-system reactivated in vitro in transporting vesicles with exogenous C-kinase and TPA or bioactive DG analogs. In cells and isolated membranes, the proteins phosphorylated by TPA- or DG-activated C-kinase will be identified by electrophoretic analysis. The proposed research is designed to increase our understanding of fundamental mechanisms of tumor promotion, including the reactivation of quiescent, differentiated cells back into a rapidly growing state. (S)
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