Bianchi Mariarita, De Lucchini Stefania, Vietri Michele, Villa-Moruzzi Emma
Department of Experimental Pathology, University of Pisa, Pisa, Italy.
Mol Cell Biochem. 2005 Apr;272(1-2):85-90. doi: 10.1007/s11010-005-7639-z.
Protein phosphatase 1delta (PP1delta) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1delta and FAK to detect their reciprocally interacting domains. Dissection of PP1delta indicated 194-260 as the shortest FAK-interacting domain among those tested. Domain 194-260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194-260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684-1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.
蛋白磷酸酶1δ（PP1δ）定位于粘着斑并与粘着斑激酶（FAK）相关联。在本研究中，我们使用PP1δ和FAK的缺失突变体来检测它们相互作用的结构域。对PP1δ的剖析表明，在所测试的结构域中，194 - 260是最短的与FAK相互作用的结构域。194 - 260结构域包含几个参与催化的位点，间接证实FAK是PP1的底物。这些位点之一R220（R220S或R220Q）的突变并没有消除，反而增强了194 - 260下拉FAK的能力。这种特性可用于检测新的潜在PP1底物。在FAK缺失突变体中，只有C末端结构域（684 - 1053，也称为FRNK）能下拉大量的PP1。当在FPLC Superose 12上通过凝胶过滤分析时，从GST - FRNK亲和柱洗脱的PP1显示分子量为35,000，表明存在分离的PP1催化亚基。
