Zhang L, Zhang Z, Long F, Lee E Y
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Biochemistry. 1996 Feb 6;35(5):1606-11. doi: 10.1021/bi9521396.
Protein phosphatase-1 (PP1) is regulated by interaction with different subunits, which include several inhibitory proteins. It is also potently inhibited by several toxins of diverse origins. Recent work has identified a region near the C-terminus of PP1 (residues 274-277) whose modification was shown to moderate okadaic acid binding [Zhang et al. (1994) J. Biol. Chem. 269, 16997-17000]. In this study, the role of this region in toxin binding was explored by site-directed mutagenesis. A residue (Tyr-272) was identified whose mutation had dramatic effects on the spectrum of inhibitor sensitivity of PP1. The IC50's of a number of mutants of Tyr-272 toward okadaic acid, tautomycin, calyculin A, microcystin-LR, nodularin, inhibitor-2, and cantharidic acid were determined and compared to that of the wild-type enzyme. The sensitivity of PP1 toward tautomycin and calyculin A was markedly decreased, by as much as 3 orders of magnitude, with lesser effects on okadaic acid and nodularin, and with microcystin-LR and inhibitor-2 being the least affected. These studies show that Tyr-272 is of specific importance for the binding of these inhibitors and provide strong evidence for the postulate that these toxins all bind to a common inhibitor site on PP1. In addition, our studies show that Tyr-272 is not required for catalytic activity.
蛋白磷酸酶-1（PP1）通过与不同亚基相互作用而受到调节，这些亚基包括几种抑制蛋白。它也受到多种来源的几种毒素的强烈抑制。最近的研究确定了PP1 C末端附近的一个区域（第274 - 277位氨基酸残基），该区域的修饰显示可调节冈田酸的结合[Zhang等人（1994年）《生物化学杂志》269卷，16997 - 17000页]。在本研究中，通过定点诱变探索了该区域在毒素结合中的作用。确定了一个残基（酪氨酸-272），其突变对PP1抑制剂敏感性谱有显著影响。测定了酪氨酸-272的多个突变体对冈田酸、互隔交链孢酚单甲醚、花萼海绵诱癌素A、微囊藻毒素-LR、节球藻毒素、抑制剂-2和斑蝥素的半数抑制浓度（IC50），并与野生型酶进行比较。PP1对互隔交链孢酚单甲醚和花萼海绵诱癌素A的敏感性显著降低，降低幅度高达3个数量级，对冈田酸和节球藻毒素的影响较小，对微囊藻毒素-LR和抑制剂-2的影响最小。这些研究表明酪氨酸-272对这些抑制剂的结合具有特定重要性，并为这些毒素都结合到PP1上一个共同的抑制剂位点这一假设提供了有力证据。此外，我们的研究表明酪氨酸-272对催化活性不是必需的。
