BACTERIAL EXPRESSION OF THE LINKER REGION OF HUMAN MDR1 P-GLYCOPROTEIN AND MUTATIONAL ANALYSIS OF PHOSPHORYLATION SITES

Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous ha lves of human P-gp harbors several serine residues which are phosphory lated by protein kinase C (PKC) in vitro. We used the glutathione S-tr ansferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the lin ker region of the human MDR1 gene product. The fusion proteins were su bjected to in vitro phosphorylation and phosphopeptide mapping analysi s to identify specific phosphorylation sites. On the basis of a mutati onal strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-66 7 were identified as major PKC sites and Ser-683 was identified as a m inor PKC site. Ser-661 and Ser-667 were also found to be the primary s ites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the muItidrug-resistant KB-V1 cell line. Individu al phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp spec ific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which o verlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applic able for the analysis of multisite and ordered protein phosphorylation in other systems.