Phosphorylation of the multidrug resistant associated glycoprotein (p-glycoprotein): Preparation and characterization of 7-acetyltaxol

Abstract

Drug-resistant cells derived from the non-mutagenized mouse macrophage-like cell line J774.2 display the multidrug resistant (MDR) phenotype which includes: (1) cross-resistance to unrelated hydrophobic drugs; (2) reduced steady-state drug accumulation and (3) the overexpression of a 130-145 kDa membrane phosphoglycoprotein (P-glycoprotein). Evidence suggests that P-glycoprotein is an energy-dependent efflux pump that maintains a reduced level of drug within the MDR cell. To assess the role of phosphorylation in P-glycoprotein function, phosphorylation of P-glycoprotein in intact cells and in cell-free membrane fractions has been studied. Results obtained with cell-free membrane fractions indicate that P-glycoprotein is a substrate for a membrane-associated protein kinase A (PK-A). To assess whether P-glycoprotein was phosphorylated in vivo by PK-A, MDR cells were incubated with ({dollar}\sp{lcub}32{rcub}{dollar}P) Pi in the presence or absence of 100 uM 8BrcAMP. The tryptic phosphopeptides of six P-glycoproteins from five independently derived MDR cell lines were analyzed by HPLC. Four of them (from J7.V1-1; J7.C1-100; J7.T2-50 and the higher MW P-glycoprotein from J7.T1-50) display a similar HPLC profile. Each contains a major phosphopeptide that was eluted with a retention time of 34 min and was resolved as two phosphopeptides by 2-D mapping. A similar analysis carried out with two other P-glycoproteins (from J7.V3-1 and the lower band of J7.T1-50) demonstrated a major phosphopeptide with a retention time of 26 min. Fraction 26 was resolved as a single phosphopeptide by 2-D mapping. The phosphorylation of fraction 26 which was derived from P-glycoprotein in J7.V3-1 or the J7.T1-50 lower band was enhanced when the cells were treated with 8BrcAMP. Our results support the idea that PK-A phosphorylates P-glycoprotein in vivo. The presence of two phosphopeptides, peaks 26 and 34, support the idea (suggested by biosynthesis and molecular biology studies) that in MDR J774.2 cells, at least two related but distinct P-glycoproteins are expressed.;Ten uM 8BrcAMP enhanced by 1.5-fold the ED{dollar}\sb{lcub}50{rcub}{dollar} for colchicine in J7.C1-100 cells. A 1.2-fold enhancement of the ED{dollar}\sb{lcub}50{rcub}{dollar} for vinblastine in J7.V3-1 was observed. These results suggest that 8BrcAMP may enhance drug resistance in MDR cells derived from J774.2. The state of phosphorylation of P-glycoprotein, which is a major component of the MDR phenotype, may be related to the role of the protein in maintaining resistance.