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.
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|