Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

Abstract

The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system. PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha. Photoaffinity labeling of membrane vesicles with [3H]azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha. In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220. Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity. These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.

Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones

“…The phosphorylation state and activity of P-gp can be augmented by PKC activators, such as tumour-promoting phorbol esters (Chambers et al, 1990(Chambers et al, , 1992Ma et al, 1991) and inhibited by agents such as staurosporine (Chambers et al, 1990(Chambers et al, , 1992Chaudhary and Roninson, 1992;Bates et al, 1993). Of the PKC isozymes, only PKC-a has to date been identified as important in the post-translational regulation of P-gp activity (Yu et al, 1991; Ahmad and Glazer, 1993;Ahmad et al, 1994). We analysed PKC expression levels and subcellular distribution to evaluate possible candidates that may have a role to play in phosphorylation and regulation of P-gp.…”

“…The sorted R123 low fluorescence cells expressed higher P-gp levels and will therefore be referred to as HP-gp (high P-gp) cells. As PKC-xc has been shown to play a role in the regulation of P-gp activity (Yu et al, 1991;Ahmad and Glazer, 1993;Ahmad et al, 1994) levels and subcellular distribution of PKC isozymes were examined. All three MCF-7 doxorubicinresistant cell lines expressed PKC-a, -C, -e and -6 ( Figure 6 and Figure 6.…”

UFA Orientalism. The “Orient” in Early German Film: Lubitsch and May

“…21 We have not only theoretical and methodological contradictions accompanied by hybrid strategies, but also a fateful obliteration in the silence around those involved -the "Orientals" -which has confronted Said"s Orientalism with the devastating charge of "Occidentalism". In other words, Said essentializes Europe and the West, the "Occident", as self-identical, fixed being which has always had an essence and a will, an imagination and a project, and the "Orient" as no more than its silenced object.…”

Protein kinases and multidrug resistance

“…Immunoprecipitation results clearly show that PKC and Pgp interact closely in the membrane and, as suggested by Bates et al (1992) and implied more recently by Germann et al (1996), phosphorylation of Pgp may influence drug substrate specificity and drug binding specificity. It may also regulate the ATPase activity of Pgp (Ahmad et al, 1994;Fine et al, 1996) perhaps accounting for the increased drug resistance seen in MDR cells which overexpress PKC. It may also be that phosphorylation by PKC has more to do with Pgp structural organisation or its normal functions (Stein, 1997) which are distinct from its role in MDR.…”