Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycine
Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycine
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DOI：
10.1074/jbc.272.21.13856
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发表时间：
1997-05-23
影响因子：
4.8
通讯作者：
Hemmings, BA
中科院分区：
文献类型：
--
作者：
Favre, B;Turowski, P;Hemmings, BA
Calyculin-A(CA)、冈田酸(OA)和平托霉素(TAU)是蛋白磷酸酶1(PP1)和蛋白磷酸酶2A(PP2A)的有效抑制剂，广泛应用于细胞培养。尽管它们在体外具有很好的选择性，但由于它们的细胞渗透特性尚不清楚，因此它们对PP1和PP2A的细胞内效应不能直接从它们的细胞外浓度来推断。在这里，我们证明，由于抑制剂与PP1和/或PP2A的紧密结合，它们的细胞渗透可以通过测量无细胞提取物中的PP1和PP2A活性来监测。10 nM CA处理MCF7细胞2 h，可同时抑制PP1和PP2A活性50%以上。在MCF7细胞中，需要1 mU的MOA才能获得与10 nM CA相似的PP2A抑制时间过程，而PP1的活性不受影响。在TAU处理的MCF7细胞中，PP1主要被抑制，但即使在10mU的TAU作用下，PP1的抑制也比10nM CA处理的慢得多。此外，Western blotting表明，在MCF7细胞中，抑制剂与PP2Ac和/或PP1c结合导致蛋白质的羧基末端发生不同的翻译后修饰。与TAU相反，OA和CA诱导PP2Ac的羧基末端Leu(309)残基去甲基化。另一方面，与OA相比，CA和TAU引起PP1c铜型异构体羧基末端的免疫反应性显著降低，这可能反映了蛋白质的蛋白降解。这些结果表明，在MCF7细胞中，OA选择性地抑制PP2A，TAU主要影响PP1，这一结论得到了它们对该细胞系中细胞角蛋白的不同影响的支持。
Calyculin-A (CA), okadaic acid (OA), and tautomycin (TAU) are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) and are widely used on cells in culture. Despite their well characterized selectivity in vitro, their exact intracellular effects on PP1 and PP2A cannot be directly deduced from their extracellular concentration because their cell permeation properties are not known. Here we demonstrate that, due to the tight binding of the inhibitors to PP1 and/or PP2A, their cell penetration could be monitored by measuring PP1 and PP2A activities in cell-free extracts. Treatment of MCF7 cells with 10 nM CA for 2 h simultaneously inhibited PP1 and PP2A activities by more than 50%. A concentration of 1 mu M OA was required to obtain a similar time course of PP2A inhibition in MCF7 cells to that observed with 10 nM CA, whereas PP1 activity was unaffected. PP1 was predominantly inhibited in MCF7 cells treated with TAU but even at 10 mu M TAU PP1 inhibition was much slower than that observed with 10 nM CA. Furthermore, binding of inhibitors to PP2Ac and/or PP1c in MCF7 cells led to differential posttranslational modifications of the carboxyl termini of the proteins as demonstrated by Western blotting. OA and CA, in contrast to TAU, induced demethylation of the carboxyl-terminal Leu(309) residue of PP2Ac. On the other hand, CA and TAU, in contrast to OA, elicited a marked decrease in immunoreactivity of the carboxyl terminus of the cu-isoform of PP1c, probably reflecting proteolysis of the protein. These results suggest that in MCF7 cells OA selectively inhibits PP2A and TAU predominantly affects PP1, a conclusion supported by their differential effects on cytokeratins in this cell line.