RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Células COS , Adesão Celular/fisiologia , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Adesões Focais/metabolismo , Humanos , Imunoprecipitação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas com Domínio LIM , Proteínas de Repetições Ricas em Leucina , Proteínas de Membrana , Camundongos , Mutação , Ligação Proteica , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Vinculina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Induction of gene expression by IL-6 has become an area of intense interest due to the role this cytokine plays in mediating aspects of inflammation, cellular differentiation, and proliferation. The ETS family of transcription factors represents a group of positive and negative regulators of transcription, that are differentially expressed in a cell and tissue specific manner. The ETS protein Fli-1 is known to induce differentiation in the erythroblastic leukemia cell line K562 along megakaryocytic developmental pathways. Here we show that IL-6 treatment of K562 induces the expression of Fli-1 via the STAT3 transcription factor. Upregulation of Fli-1 expression can be abrogated by the addition of AG490, a chemical inhibitor of JAK kinases, and by transfecting the cells with a dominant negative STAT3 expression construct.
