PHOSPHORYLATION BY PROTEIN-KINASE-C AND CYCLIC-AMP-DEPENDENT PROTEIN-KINASE OF SYNTHETIC PEPTIDES DERIVED FROM THE LINKER REGION OF HUMAN P-GLYCOPROTEIN

Specific sites in the linker region of human P-glycoprotein phosphoryl ated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type reco gnized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PK C in a Ca2+/phospholipid-dependent manner, with a K-m of 1.3 mu M, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of pep tide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PE;C identified Ser-661, Ser-667 and Ser-671 as the three sites of ph osphorylation. PG-2 was also found to be phosphorylated by purified PK A in a cyclic AMP-dependent manner, with a K-m of 21 mu M, and to a ma ximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Se r-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated pepti des of PG-2, were utilized to confirm that Ser-661 was PKC-specific an d Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membr anes from multidrug-resistant human KB cells. The membrane kinase phos phorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two p rotein kinases, stimulated by different second-messenger systems, whic h exhibit both overlapping and unique specificities for phosphorylatio n of multiple sites in the linker region of the molecule.