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 32P-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 anti-sera against P-glycoprotein suggests that P35 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.
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Division of Biology, Kansas State University, Manhattan 66506.
Marquardt
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Takemoto
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Center
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002460:Cell Line; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates / Q000737:chemistry; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D019311:Staurosporine; D014357:Trypsin / Q000737:chemistry; D014750:Vincristine / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000470:Alkaloids; D000596:Amino Acids; D005561:Formates; D008562:Membrane Glycoproteins; C030544:formic acid; D014750:Vincristine; D014357:Trypsin; D019311:Staurosporine