From screening to function - Evolutionary conservation of novel JAK/STAT signal transduction pathway components

Abstract

Signal transduction pathways mediating the exchange of information between cells are essential for development, cellular differentiation and homeostasis. Their dysregulation is also frequently associated with human malignancies. The Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) cascade represents one such signaling pathway whose evolutionarily conserved roles include cell proliferation and hematopoiesis. In the present study, a systematic genome-wide screen for genes required for JAK/STAT pathway activity has been performed. By analyzing 20,026 RNA interference (RNAi)-induced phenotypes in cultured Drosophila melanogaster hemocyte-like cells, a total of four previously known and 86 novel and uncharacterized genes were discovered. Subsequently, cell-based epistasis experiments were used to classify these modulators based on their interaction with known components of the signaling cascade. To analyze the functional conservation of these novel components throughout evolution, putative human homologs of the candidates found in Drosophila were targeted in human cell culture systems to assess the activity of human STATs upon knockdown of candidate modulators. Interestingly, 30 of the human homologs display a similar JAK/STAT phenotype to their Drosophila counterparts. In addition to multiple human disease gene homologs, the protein tyrosine phosphatase Ptp61F and the Drosophila homolog of BRWD3, a bromo-domain-containing protein disrupted in leukemia, were found in the RNAi screen. In vivo analysis of these two novel pathway regulators demonstrates that disrupted dBRWD3 and overexpressed ptp61F function as suppressors of leukemia-like blood cell tumors. Moreover, dBRWD3 is localized in the nucleus and can physically interact with Drosophila STAT, likely to induce target gene activity. The present study represents a comprehensive identification of novel loci required for JAK/STAT signaling and provides molecular insights into an important pathway relevant for human cancer. Human homologs of identified pathway modifiers may constitute targets for therapeutic interventions.