DeFranco D B, Qi M, Borror K C, Garabedian M J, Brautigan D L
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Mol Endocrinol. 1991 Sep;5(9):1215-28. doi: 10.1210/mend-5-9-1215.
We have used okadaic acid (OA), a cell-permeable inhibitor of serine/threonine protein phosphatase types 1 (PP-1) and 2A (PP-2A), to demonstrate that the subcellular distribution of glucocorticoid receptor (GR) in rat fibroblasts is influenced by its phosphorylation state. Nuclear GRs in OA-treated cells retain transcriptional enhancement activity. Nuclear import or export of hormone agonist-bound GRs is not affected by OA. However, a dose of OA that fully inhibits PP-2A and partially inhibits PP-1, but not a lower dose that only partially inhibits PP-2A, leads to inefficient nuclear retention of agonist-bound GRs, and their redistribution into the cytoplasm. These receptors appear to be trapped in the cytoplasmic compartment and are unable to recycle (i.e. reenter the nucleus). Addition of OA during different steps of GR recycling demonstrates that OA must be present during nuclear export of GRs to block GR recycling. A direct role for PP-1 and/or PP-2A in GR recycling is suggested by site-specific hyperphosphorylation of GRs in vivo during OA inhibition of recycling. These are the same sites that undergo in vitro site-specific dephosphorylation by PP-1 and PP-2A. The block in GR recycling that results from inhibition of PP-1 and/or PP-2A resembles effects previously observed in v-mos-transformed rat fibroblasts. Interestingly, OA inhibition of PP-2A in v-mos-transformed cells leads to the reversal of oncoprotein effects on GR recycling and retention of receptors within the nuclear compartment. We propose that GR recycling is influenced by the activities of distinct protein phosphatases (PP-1 and/or PP-2A), and that the interference of this pathway observed in v-mos-transformed cells may be the result of effects of the oncoprotein on the phosphatases or a specific subset of their targets.
我们使用了冈田酸（OA），一种可穿透细胞的丝氨酸/苏氨酸蛋白磷酸酶1型（PP - 1）和2A 型（PP - 2A）抑制剂，来证明糖皮质激素受体（GR）在大鼠成纤维细胞中的亚细胞分布受其磷酸化状态影响。OA处理细胞中的核GR保留转录增强活性。激素激动剂结合的GR的核输入或输出不受OA影响。然而，能完全抑制PP - 2A并部分抑制PP - 1的OA剂量，而非仅部分抑制PP - 2A的较低剂量，会导致激动剂结合的GR在核内保留效率低下，并重新分布到细胞质中。这些受体似乎被困在细胞质区室中，无法再循环（即重新进入细胞核）。在GR再循环的不同步骤中添加OA表明，GR核输出过程中必须存在OA才能阻断GR再循环。OA抑制再循环过程中体内GR的位点特异性过度磷酸化提示了PP - 1和/或PP - 2A在GR再循环中的直接作用。这些位点正是体外被PP - 1和PP - 2A进行位点特异性去磷酸化的位点。PP - 1和/或PP - 2A抑制导致的GR再循环阻断类似于先前在v - mos转化的大鼠成纤维细胞中观察到的效应。有趣的是，OA对v - mos转化细胞中PP - 2A的抑制导致癌蛋白对GR再循环作用的逆转以及受体在核区室中的保留。我们提出GR再循环受不同蛋白磷酸酶（PP - 1和/或PP - 2A）活性的影响，并且在v - mos转化细胞中观察到的该途径的干扰可能是癌蛋白对磷酸酶或其特定目标子集作用的结果。
