Characterization of Phosphorylation-defective Mutants of Human P-glycoprotein Expressed in Mammalian Cells

Abstract

To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [ 32 P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glycoprotein-specific protein kinase purified from multidrugresistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.

Structural and Functional Consequences of Mutating Cysteine Residues in the Amino Terminus of Human Multidrug Resistance-associated Protein 1

“…It has been shown previously that MRP1 has at least two drug-binding sites (35) and that these putative drug-binding sites are likely located in the transmembrane segments 10 and 11 in MSD2 and segments 16 and 17 in MSD3 (29,36), similar to that found in P-glycoprotein (37)(38)(39). The MSD1 domain including the putative amino terminus is not labeled by photo affinity substrates, and its absence does not affect LTC 4 labeling of MRP1 (36).…”

Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo

“…On the other hand, experiments with artificially introduced MDR-1 mutations show clearly that PGP reacts quite sensitively to amino acid alterations. Mutations that change amino acids can alter the substrate spectrum of PGP, the effectiveness of transport, and also the sensitivity of PGP toward inhibition with specific inhibitory substances (2,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). It is clear that some naturally occurring mutations and alleles-if they exist, and if we can discover themmay show similar effects.…”

Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC η with MDR1, MRP and LRP gene expression

“…An influence of protein kinase C (PKC) on P-gp-mediated MDR is suggested as well, but it still remains unclear how PKC or particular PKC isozymes might be functionally involved here: (a) in vitro studies on cell lines gave evidence that P-gp is directly phosphorylated by PKC (Chambers et al, 1990a,b); (b) co-overexpression of PKCa pointed to a possible role of this PKC isozyme in MDR of breast cancer cell lines (Yu et al, 1991); (c) MRPmediated MDR was significantly modulated by the specific PKC inhibitor GO 6850 ; but (d) PKC-mediated phosphorylation of P-gp seems not to be significantly associated with altered P-gp drug transport function (Scala et al, 1995;Gekeler et al, 1996;Germann et al, 1996). Remarkably, the induction of a P-gp-mediated MDR in cell lines using different anticancer drugs (Gekeler et al, 1988;Sato et al, 1990) could be attenuated by using protein kinase inhibitors such as staurosporine, which suggested a protein kinase-triggered stress response of cells including the up-regulation at least of the human MDR1 gene (Chaudhary and Roninson, 1993;Blobe et al, 1994;Grunicke et al, 1994).…”