Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells
Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells
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DOI：
10.1074/jbc.271.3.1708
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发表时间：
1996-01-19
影响因子：
4.8
通讯作者：
Gottesman, MM
中科院分区：
文献类型：
--
作者：
Germann, UA;Chambers, TC;Gottesman, MM
为了评估人类多药耐药基因产物P-糖蛋白的磷酸化对其药物转运活性的作用，对其连接区的磷酸化位点进行了突变分析。我们构建了一个5A突变体，其中661、667、671、675和683位的丝氨酸被非磷酸化的丙氨酸残基取代，以及一个在相应位置携带天冬氨酸残基的5D突变体，以模拟永久磷酸化的丝氨酸残基。转染研究表明，这两个突变体都能正确地定位于细胞表面，并通过减少药物积累而产生多药耐药。与野生型P-糖蛋白相比，过表达的5A和5D突变体没有检测到磷酸化水平，无论是在体内用[P-32]正磷酸盐标记细胞，还是在体外用蛋白激酶C、cAMP依赖的蛋白激酶或从多药耐药KB-V1细胞中纯化的P-糖蛋白特异性蛋白激酶进行磷酸化检测。这些结果再次证实了P-糖蛋白的主要磷酸化位点位于连接区。此外，首次提供的直接证据表明，磷酸化/去磷酸化机制在人P-糖蛋白介导的多药耐药表型的建立中不起重要作用。
To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [P-32] orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glycoprotein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.