Bates S E, Lee J S, Dickstein B, Spolyar M, Fojo A T
Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
Biochemistry. 1993 Sep 7;32(35):9156-64. doi: 10.1021/bi00086a022.
Previous studies of P-glycoprotein have demonstrated that its function can be modulated by phosphorylation. In the present study, inhibition of protein kinase C with calphostin C or stauroporine or prolonged treatment with the phorbol ester TPA decreased phosphorylation of P-glycoprotein, and impaired transport of vinblastine. Calphostin C also inhibited transport of actinomycin D, vincristine, rhodamine, and azidopine in SW620 Ad300 multidrug-resistant human colon carcinoma cells. Photoaffinity labeling of P-glycoprotein with azidopine was decreased by calphostin C, suggesting that dephosphorylation alters the affinity of P-glycoprotein for its substrates. Impaired transport of rhodamine in normal T lymphocytes treated with staurosporine demonstrates that modulation of P-glycoprotein function is not limited to cells selected for drug resistance in vitro. Transport of P-glycoprotein antagonists in SW620 Ad300 cells was also affected by calphostin C. Cyclosporin A transport decreased, while verapamil transport increased. Cyclosporin A in calphostin C-treated cells resulted in additive P-glycoprotein antagonism, while no additive effect could be demonstrated with verapamil, suggesting that the increase in verapamil transport makes it a poorer P-glycoprotein antagonist. These studies suggest that transport by P-glycoprotein is a dynamic process which can be modulated by phosphorylation, and that antagonists may block P-glycoprotein differently in different phosphorylation states.
先前对P-糖蛋白的研究表明，其功能可通过磷酸化作用进行调节。在本研究中，用钙泊三醇C或星形孢菌素抑制蛋白激酶C，或用佛波酯TPA进行长时间处理，均可降低P-糖蛋白的磷酸化水平，并损害长春碱的转运。钙泊三醇C还可抑制SW620 Ad300多药耐药人结肠癌细胞中放线菌素D、长春新碱、罗丹明和叠氮平的转运。钙泊三醇C可降低叠氮平对P-糖蛋白的光亲和标记，提示去磷酸化改变了P-糖蛋白对其底物的亲和力。用星形孢菌素处理的正常T淋巴细胞中罗丹明转运受损，表明P-糖蛋白功能的调节并不局限于体外选择的耐药细胞。钙泊三醇C也影响SW620 Ad300细胞中P-糖蛋白拮抗剂的转运。环孢素A的转运减少，而维拉帕米的转运增加。在钙泊三醇C处理的细胞中，环孢素A导致P-糖蛋白拮抗作用增强，而维拉帕米未显示出增强作用，提示维拉帕米转运的增加使其成为较差的P-糖蛋白拮抗剂。这些研究表明，P-糖蛋白介导的转运是一个动态过程，可通过磷酸化作用进行调节，并且拮抗剂在不同的磷酸化状态下可能对P-糖蛋白产生不同的阻断作用。
