Intracellular distribution and regulatory function of protein phosphatases

在天然存在的1型和2A型蛋白磷酸酶抑制剂(PP1和PP2A)中，最早从螺旋链霉菌中分离出来的图图霉素(TM)是独一无二的，因为它与PP1结合并抑制PP1的亲和力高于PP2A。PP1-TM相互作用的离解常数与PP2A-TM相互作用的离解常数之比(PP1/PP2A之比)在0.01-0.03范围内。到目前为止，还没有确定导致其与酶的特征亲和力的特定结构因素。在本实验中，我们评估了TM的C1-C16片段对其与PP1和PPAA结合的贡献。根据目前的结合模型，这一相对疏水的片段包含一个带有两个侧链的螺酮基序，应该被调节在离酶的催化中心略远的位置。因此，我们预计这一片段的化学修饰可能会产生一些衍生的…更多的S保留了可测的抑制活性。我们还特别感兴趣的是，螺环酮的对映体形式存在于OA分子中，与TM相反，它对PP2A的亲和力比对PP1高得多。由于有许多大分子与相对较小的配体的立体特异性相互作用的例子，似乎很自然地推测螺酮的立体化学可能是决定毒素亲和力特性的关键因素。出于这些原因，我们化学合成了两个TM类似物：TM1，其中螺酮的侧链被移除，但其立体化学保持不变；以及TM2，其中TM1的螺酮基序被其对映体形式取代。我们观察到这些衍生物确实对PP1和PP2A具有相当大的抑制活性。通过使用天然催化亚基PP1(PP1C)和PP2A以及重组PP1的γ异构体(PP1TM2)进行剂量抑制分析，确定的PP1/PP2A比率在TM1的0.20-0.5%和TM2的5-10%范围内。TM与TM1和TM2的衍生化所引起的比值显著增加，表明螺酮的立体化学及其侧链的存在是TM与PP1和PP2A特征亲和力的重要因素。较少

Among the naturally occurring inhibitors of type 1 and type 2A protein phosphatases (PP1 and PP2A), tatutomycin (TM), first isolated from the bacterium Streptomyces spiroverticillatus, is unique in that it binds to and inhibits PP1 with higher affinity than it does PP2A . The ratio of the dissociation constant for the PP1-TM interaction to that for the PP2A-TM interaction (the PP1/PP2A ratio) is in the range 0.01-0.03. No specific structural factors responsible for its characteristic affinity to the enzymes have been identified until now.In the present experiments we have evaluated the contributions of the C1-C16 segment of TM to its binding to PP1 and PP2A.According to the current binding model, this relatively hydrophobic segment, which contains a spiroketal motif with two side chains, are supposed to be accommodated in a site which is slightly apart from the catalytic center of the enzymes. We therefore expected that chemical modifications of this segment might yield some derivative … More s retaining a measurable inhibitory activity. We were also particularly interested in the fact that the enantiomeric form of the spiroketal is present in the molecule of OA, which, in contrast to TM, exhibits exceedingly higher affinity to PP2A than to PP1. Since there are numerous examples of stereospecific interaction of a macromolecule with a relatively small ligand, it seemed natural to speculate that the stereochemistry of the spiroketal might be a key factor determining the affinity characteristics of the toxins.For these reasons we have chemically synthesized two TM analogues : TM1 in which the side chains of the spiroketal is removed but its stereochemistry is retained, and TM2 in which the spiroketal motif of TM1 is replaced with its enantiomeric form. We have observed that these derivatives indeed retain considerable inhibitory activities against PP1 and PP2A.The PP1/PP2A ratio, determined by dose-inhibition analyses using the native catalytic subunits of PP1 (PP1C) and PP2A and a recombinant γ isoform of PP1(PP1γ), is in the range 0.2-0.5 with TM1 and 5-10 with TM2. The marked increases in the ratio caused by the derivatization of TM to TM1 and TM2 indicate that the stereochemistry of the spiroketal as well as the presence of its side chains is an important factor for the characteristic affinities of TM to PP1 and PP2A. Less