P-glycoprotein (Pgp) is an ATPase thought to extrude actively anticancer drugs from the cells. Overexpression of Pgp seems to be a frequent cause of failure of chemotherapy in the treatment of some forms of cancer. Pgp is also expressed in normal tissues including adrenal cortex, proximal renal tubules of the kidney and endothelial cells, where it appears to be involved in secretion of aldosterone and transport of drugs and metabolites, respectively. It seems that phosphorylation of Pgp by protein kinases A (PKA) and C (PKC) activates drug transport, and it has been shown that PKC activity is elevated in several multidrug-resistant cell lines. Apparently, PKA and PKC phosphorylate only 3 serines each (two overlapping), which are located in the linker region between the two homologous halves of Pgp (minilinker domain). The main objectives of this proposal are to understand the mechanisms by which PKA- and PKC-mediated phosphorylation of Pgp affect to transport of organic substrates, and to define the basic mechanisms of regulation of Pgp activity via changes in PKC activity. To accomplish these aims, we will study the effects of Pgp phosphorylation of PKA and PKC on drug transport and binding, and ATP binding and hydrolysis. We will carry out studies 'in vivo' on intact cells and 'In vitro' using full- length Pgp and Pgp fragments that contain the N-terminal ATP- and drug-binding domains and the minilinker domain. In the intact cells, we will identify the functionally relevant serines by determining the effects of phosphorylation on cells expressing wild-type and mutant Pgps. In the mutant Pgps, serines of the minilinker domain will be substituted by alanines, individually or in combinations. Finally, we will use a Pgp mutant that does not respond to phosphorylation by PKC to assess the role of the kinase in the stimulation of Pgp in response to exposure to cytotoxic agents, estradiol, and the antiestrogen tamoxifen. The understanding of the molecular bases of the effects of PKA and PKC on Pgp-mediated drug transport will be an important contribution to our knowledge of the regulation of Pgp function. The experiments in this proposal will also provide the framework for studies aimed to regulate Pgp function and multidrug resistance by low-toxicity agents (e.g., endogenous hormones).
