title: The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions.
abstract: 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.
authors.affiliation: Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, B3122, Bethesda, MD 20814, USA.
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Dougherty
authors.forename: Treasa
authors.initials: T
authors.lastname: Chopp
authors.forename: Sheng-Mei
authors.initials: SM
authors.lastname: Qi
authors.forename: Mary Lou
authors.initials: ML
authors.lastname: Cutler
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D060588:LIM Domain Proteins; D000090263:Leucine-Rich Repeat Proteins; D008565:Membrane Proteins; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016596:Vinculin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; D000090263:Leucine-Rich Repeat Proteins; C490232:Lims1 protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D034741:RNA, Small Interfering; C484099:Rsu1 protein, mouse; D014157:Transcription Factors; D016596:Vinculin; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases