Pgp and protein kinase C (Castro, 1999). Pgp activity (like everything else in the cell) does not occur independently of signal transduction pathways; in this case, Pgp activity is connected to protein kinase C (PKC). PKC phosphorylates Pgp and overstimulation of PKC results in increased Pgp-mediated substrate movement. Similarly, PKC inhibitors (staurosporine and chelerythrine) result in a build-up of toxic chemicals in MDR cells expressing Pgp, in this case the cancer cell line MCF-7 (Figure 25). This seems to indicate that PKC-induced phosphorylation drives Pgp transport. However, PKC inhibitors are also known to interact directly with the Pgp protein, thus raising additional questions as to the actual mechanism of PKC and Pgp activity. Once again, analysis of Pgp mutants has proven useful in the study of mechanistic details.

Figure 25 (figure taken from Castro, 1999)

PKC mechanistic complexities (Castro, 1999). In studies of MDR breast cancer cells, both the general PKC blocker staurosporine and the more specific chererythrine resulted in greater accumulation of toxic chemicals within the cell (Figure 25). However, this data confirms previously established facts but provides no new insight into mechanistic details. As a result, the time course with which PKC blockers inhibit Pgp was measured. Phosphorylation/dephosphorylation would require approximately 30 minutes to take effect, while direct interaction between the PKC inhibitor and Pgp would be much faster. Upon treatment with staurosporine, a general PKC inhibitor, Pgp inhibition became measurable after approximately 2 minutes, supporting the postulation that PKC and PKC inhibitors have a more far-reaching effect upon Pgp than a simple activation/inhibition relationship (Figure 26). Figure 26A demonstrates the drastic decrease in Pgp transport ability following treatment with a PKC blocker.

Figure 26 (figure taken from Castro, 1999)

Pgp mutants and phosphorylation (Castro, 1999). To gather further evidence of the importance of phosphorylation and Pgp activity, PKC inhibition experiments were repeated using both wild-type cells and cells containing Pgp mutants. The mutant Pgp was no longer capable of being phosphorylated due to replacement of Ser residues with Ala residues. Rate constants for drug transport were directly related to the degree of Pgp expression in both mutants and normal cells. As a result, although phosphorylation may be involved in activating Pgp, it is not required for function. The loss of phosphorylation sites is not enough to impact transport. In further experiments, the Pgp mutant proteins behaved exactly like the wild-type Pgp upon exposure to cytoxic agents thus furthering the conclusion that phosphorylation sites are not absolutely required for the expression of MDR. Both the wild-type cells as well as the Ser-Ala replacement cells were equally inhibited by exposure to a PKC blocker (Figure 26B). The rapid effect of PKC blockers upon drug transport appears to be independent of Pgp phosphorylation. These results are likely due to direct competition between Pgp substrate and the inhibitors for Pgp biding sites. In addition, it is possible that the PKC inhibitor actually prevents the phosphorylation of a regulatory protein which then influences Pgp.