ANALYSIS OF P-GLYCOPROTEIN PHOSPHORYLATION IN HL60 CELLS ISOLATED FOR RESISTANCE TO VINCRISTINE

In the present study we have analyzed the involvement of phosphorylation in the function of P-glycoprotein and have also examined sites of phosphorylation along the P-glycoprotein polypeptide chain. The results show that in HL60 cells isolated for resistance to vincristine the protein kinase inhibitor staurosporine induces a major inhibition in the phosphorylation of P-glycoprotein. Further studies show that under the same conditions in which staurosporine inhibits P-glycoprotein phosphorylation there is a concomitant increase in cellular drug accumulation and a major inhibition in drug efflux. Additional studies using pulse-chase experiments show that the P-glycoprotein phosphate groups are metabolically active and that the protein undergoes rapid cycles of phosphorylation and dephosphorylation in the cell. Structural analyses demonstrate that cleavage of P-32-labeled P-glycoprotein at Asp-Pro linkages with formic acid results in the formation of a major phosphorylated peptide of 35 kDa and a minor peptide of 42 kDa. Western blot analysis using site-specific antisera against P-glycoprotein suggests that P-35 represents a phosphorylated fragment containing P-glycoprotein amino acids 446-744. Analysis of tryptic peptides using site-specific antisera identifies a second major phosphorylated region of P-glycoprotein which contains amino acids 745-1088. These studies thus suggest that phosphorylation plays an important role in the biological activity of P-glycoprotein. The results also indicate that two adjacent internal regions are highly phosphorylated in the P-glycoprotein molecule.