The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

• Published in: Experimental cell research 2005-05, Vol.306 (1), p.168-179
• Main Authors: Dougherty, Gerard W., Chopp, Treasa, Qi, Sheng-mei, Cutler, Mary Lou
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; Adaptor Proteins, Signal Transducing; Animals; Binding Sites; BIOLOGICAL STRESS; Cell Adhesion - physiology; Cell Line; Cercopithecus aethiops; COS Cells; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Focal Adhesions - metabolism; GENE REGULATION; GLUTATHIONE; GROWTH; Humans; HYBRIDIZATION; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases - metabolism; Jun kinase; LEUCINE; Leucine rich repeat; LIM domain; LIM Domain Proteins; Membrane Proteins; Mice; Mutation; p38 Mitogen-Activated Protein Kinases - metabolism; PHOSPHOTRANSFERASES; PINCH; Protein Binding; Proteins - metabolism; Ras; RNA; RNA, Small Interfering - genetics; Rsu-1; Saccharomyces cerevisiae - genetics; Transcription Factors - genetics; Transcription Factors - metabolism; Transfection; TUMOR CELLS; Two-Hybrid System Techniques; Vinculin - metabolism; YEASTS
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2005.01.025

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.