Lelong I H, Cardarelli C O, Gottesman M M, Pastan I
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1994 Aug 2;33(30):8921-9. doi: 10.1021/bi00196a009.
We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.
我们之前已经表明，在从多药耐药细胞系KB-V1分离的质膜囊泡中，鸟苷三磷酸（GTP）可以替代三磷酸腺苷（ATP）作为能量来源，以支持多药转运蛋白P-糖蛋白（Pgp）转运长春碱[勒隆等人（1992年）《欧洲生物化学学会联合会快报》304卷，第256 - 260页]。与[γ-32P]ATP一样，[γ-32P]GTP在体外也能够使Pgp磷酸化。未标记的GTP增强了[γ-32P]ATP对转运蛋白的磷酸化作用，而未标记的ATP则抑制标记物的掺入。虽然[γ-32P]ATP的磷酸化作用依赖于Mg(2+)，但GTP对Pgp的磷酸化增强作用由Mg2+或Mn2+支持，在较小程度上也由Ca2+支持。环磷酸腺苷（cAMP）依赖性蛋白激酶、蛋白激酶C和环磷酸鸟苷（cGMP）依赖性蛋白激酶的特异性抑制剂不影响磷酸化作用。磷蛋白磷酸酶抑制剂冈田酸略微增强了磷酸化作用，而钒酸盐则更显著地增加了转运蛋白的磷酸化作用。Pgp磷酸化肽段的胰蛋白酶图谱表明，添加GTP改变了磷酸肽的相对标记情况。这些结果表明，体外Pgp的总体磷酸化作用由几种不同的蛋白激酶和磷酸酶决定，其中至少一种可能受GTP调节。
