Positive feedback between transcriptional and kinase suppression accompanies extraordinary longevity and stress resistance to Caenorhabditis elegans

Insulin/IGF signaling (IIS) is conserved from yeast to mammals. Mutations conditionally disrupting either daf-2, encoding an insulinlike receptor, or age-1 encoding PI3KCS, arrest development of larvae or extend lifespan of adults at permissive temperature. This pathway negatively regulates FOXO transcription factor DAF-16 and limits longevity. Mutations that attenuate signal transduction within this pathway reduce phosphorylation of DAF-16, which when unphosphorylated enters the nucleus to modulate transcription of several hundred target genes. The first generation (F1) of kinase null allele of age-1(mg44) has 2.2--2.6 extented lifespan compare to the wild type N2DRM. The progeny of the Fl homozygous age-1(mg44) has long believed develop only as dauers. However, our lab recently reported that slowly mature at 15° or 20°C into adults with ∼10-fold extended life span. This is the greatest lifespan extension with a single gene mutation. Here we report >90% loss of kinase activity in vitro, and >40% reduction in phosphoproteins produced in vivo, F2 age-1(mg44) homozygotes, lacking all PI3K kinase activity. The kinase activity is partially restored with addition of a second mutation to daf-16 (either the m26 or mu86 allele) whereas same mutation results in nearly complete restoration of wild-type in vivo phosphoprotein levels. The unexpected effects of DAF-16 on these phenotypes lead us to determine transcript levels for a set of signal transduction molecules. IIS was previously believed to proceed by a succession of kinase-catalysed phosphorylations. Our results demonstrate for the first time the involvement of a transcriptional positive-feedback loop, in which kinase insufficiency leads to transcriptional suppression of almost all IIS components, even upstream of P13K, as well as many signaling molecules either known or not previously known to cross-talk with IIS. In addition, based on microarrays comparing F2 age-1(mg44) homozygotes to the wild type N2DRM, age-1 (hx546) and double mutants age-1(mg44; daf-16(m26), we identified 80 genes which were later quantified by real time PCR. We identified many genes belonging to distinct families such as lipid, sugar, RNA, ubiquitin metabolism genes exclusively modulated by F2 age-1 (mg44) homozygotes. We also identified two novel genes lbp-6 and F53H1.3 which extends lifespan 33 and 68% respectively.