To investigate additional components that may regulate the
daf-2/insulin-like signaling pathway, we performed a small scale RNAi screen focusing on putative signal transduction molecules. We found that RNAi inactivation of
ftt-2 enhanced the dauer formation phenotype of the
daf-2(
e1370) temperature sensitive mutant at the semi-restrictive temperature. In addition, RNAi knock down of
ftt-2 stimulated DAF-16::GFP nuclear accumulation.
ftt-2 encodes a 14-3-3 protein. The 14-3-3 family of proteins are structurally and functionally highly conserved. In contrast to
ftt-2, RNAi inactivation of
par-5/ftt-1, the only other 14-3-3 protein in C. elegans, did not show notable effects on dauer formation phenotype or DAF-16::GFP, suggesting that FTT-2 specifically regulates the
daf-2/insulin-like signaling pathway. Mammalian 14-3-3 has previously been shown to bind to DAF-16 in vitro (1), and in mammalian cells, 14-3-3 sequesters the DAF-16 orthologs FOXOs in the cytoplasm (2). Therefore, our results suggest that in C. elegans, FTT-2 also regulates
daf-2/insulin-like signaling by sequestering the forkhead transcription factor DAF-16 from the nucleus. Interestingly, RNAi knock down of
ftt-2 also resulted in dramatic decrease of DAF-16 expression levels. We demonstrated that
ftt-2 RNAi specifically affected DAF-16 protein levels, but not
daf-16 RNA levels. Considering the recent findings in mammalian cells indicating that the FOXOs proteins are targeted for polyubiquitin-mediated protein degradation, we propose that, in C. elegans, the regulation of DAF-16 levels by FTT-2 is likely through ubiquitin/proteasome degradation. Therefore, in C. elegans, FTT-2 is able to regulate DAF-16 via cytoplasmic sequestration, as well as protection from protein degradation. References: 1) Catherine M. Cahill, Guri Tzivion, Nargis Nasrin, Scott Ogg, Justin Dore, Gary Ruvkun, and Maria Alexander-Bridges. Phosphatidylinositol 3-Kinase Signaling Inhibits DAF-16 DNA Binding and Function via 14-3-3-dependent and 14-3-3-independent Pathways. J. Biol. Chem., Vol. 276, 13402-13410, 2001 2) Anne Brunet, Fumihiko Kanai, Justine Stehn, Jian Xu, Dilara Sarbassova, John V. Frangioni, Sorab N. Dalal, James A. DeCaprio, Michael E. Greenberg and Michael B. Yaffe. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. The Journal of Cell Biology, Vol. 156, 817-828, 2002