Cuppen E, Gerrits H, Pepers B, Wieringa B, Hendriks W
Department of Cell Biology and Histology, Institute of Cellular Signaling, University of Nijmegen, the Netherlands.
Mol Biol Cell. 1998 Mar;9(3):671-83. doi: 10.1091/mbc.9.3.671.
The specificity of protein-protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.
细胞信号级联反应中蛋白质-蛋白质相互作用的特异性取决于不同氨基酸基序的序列和分子内位置。我们利用双杂交相互作用陷阱来鉴定能够与蛋白酪氨酸磷酸酶PTP-BL中富含PDZ基序的片段相互作用的蛋白质。发现它与含有Lin-11、Isl-1、Mec-3（LIM）结构域的蛋白质RIL存在特异性相互作用。更详细的分析表明，结合特异性存在于PTP-BL的第二个和第四个PDZ基序以及RIL的LIM结构域中。对各种小鼠组织进行的免疫组织化学分析显示，PTP-BL和RIL在上皮细胞的膜下共定位。值得注意的是，RIL本身也有一个N端PDZ基序，它可以与RIL-LIM结构域结合。我们在此证明，RIL-LIM结构域在体外和体内均可被酪氨酸磷酸化，并且在体外可被PTP-BL的PTPase结构域去磷酸化。我们的数据表明RIL-LIM结构域上存在双PDZ结合界面，并提示酪氨酸磷酸化是多蛋白复合物组装过程中LIM-PDZ关联的一种调节机制。这些发现与PDZ介导的相互作用在极化细胞的膜下微环境的形成和组织中的重要作用一致。
