CHARACTERIZATION OF PHOSPHORYLATION-DEFECTIVE MUTANTS OF HUMAN P-GLYCOPROTEIN EXPRESSED IN MAMMALIAN-CELLS

To assess the role of phosphorylation of the human multidrug resistanc e MDR1 gene product P-glycoprotein for its drug transport activity, ph osphorylation sites within its linker region were subjected to mutatio nal analysis. We constructed a 5A mutant, in which serines at position s 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable ala nine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residu es. Transfection studies revealed that both mutants were targeted prop erly to the cell surface and conferred multidrug resistance by diminis hing drug accumulation. In contrast to wild-type P-glycoprotein, the o verexpressed 5A and the 5D mutants exhibited no detectable levels of p hosphorylation, either in vivo following metabolic labeling of cells w ith [P-32] orthophosphate or in vitro in phosphorylation assays with p rotein kinase C, cAMP-dependent protein kinase, or a P-glycoprotein-sp ecific protein kinase purified from multidrug-resistant KB-V1 cells. T hese results reconfirm that the major P-glycoprotein phosphorylation s ites are located within the linker region. Furthermore, the first dire ct evidence is provided that phosphorylation/dephosphorylation mechani sms do not play an essential role in the establishment of the multidru g resistance phenotype mediated by human P-glycoprotein.