Differential phosphorylation of sites in the linker region of P-glycoprotein by protein kinase C isozymes alpha, beta I, beta II, gamma, delta, epsilon, eta, and zeta

To determine whether individual protein kinase C (PKC) isozymes differentia lly phosphorylate sites in the linker region of human P-glycoprotein (P-gp) , we used a synthetic peptide substrate, PG-2, exactly corresponding to ami no acid residues spanning the region 656-689 of the multidrug resistance ge ne (MDR1). All tested PKC isozymes phosphorylated PG-2. The maximum phospha te incorporation by calcium-dependent PKC isozymes alpha, beta I, beta II, and gamma was 3, 2, 2, and 3 mol phosphate/mol PG-2, respectively. The maxi mum phosphate incorporation by calcium-independent isozymes delta, epsilon, eta, and zeta was 1.5, 0.5, 1.5, and 1.5 mol phosphate/mol PG-2, respectiv ely. Two-dimensional tryptic phosphopeptide mapping indicated differential phosphorylation of the PKC consensus sites Ser-661, Ser-667, and Ser-671 by individual isozymes, which may be functionally significant. These data sug gest that differential phosphorylation by PKC isoenzymes of PKC sites withi n the P-gp linker region may play a role in modulating P-gp activity. (C) 1 999 Elsevier Science Inc.