The purpose of this project is to characterize protein phosphorylation and the associated Ca2+- and phospholipid-dependent protein kinase C (PKC) activities in multidrug resistant (mdr) cells. The overexpression of PKC is closely associated with the mdr phenotype in both leukemic and breast carcinoma cell lines. Since PKC is a family of enzymes with strikingly different responses to proteolysis and phorbol ester activation, the first goal of this proposal will be to determine the relative abundance of the three major isoforms of PKC in mdr cells in comparison to sensitive cells using chromatographic and protein blotting techniques. Secondly, mdr cells selectively activate proteolytically, PKC to the Ca2+- and phospholipid-independent form termed protein kinase M (PKM). Therefore, the second goal of this study is to quantitate the levels and isoforms of PKM in mdr cells. The generation of PKM in mdr cells results in the utilization of the actin binding cytoskeletal protein, vinculin, as an endogenous substrate. Therefore, the third goal of this study is to assess vinculin phosphorylation both in vitro and in vivo. In a similar context, the last objective of this project is to assess the phosphorylation of the mdr-associated P-glycoproteins. This will be accomplished by determining: 1) whether specific antibodies to the isoforms of PKC block phosphorylation of P-glycoproteins in vitro. 2) whether phosphorylation of P-glycoproteins can be modulated in vivo or in vivo by TPA and 3) whether the use of inhibitors of PKC such as K252b block the phosphorylation of P- glycoproteins and the drug resistance-associated increases in drug binding and drug efflux.
