Abstract

Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells. This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation. In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid. Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro. The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells. Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of [3H]vinblastine. These compounds did not significantly alter [3H]vinblastine levels in drug-sensitive KB-3 cells. These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity.