Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling.
BACKGROUND
In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals.


RESULTS
This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes.


CONCLUSIONS
A screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants.
Gami
Minaxi S
MS
Laboratory of Neurosciences, NIA, NIH, Baltimore, MD, USA. gamimi@grc.nia.nih.gov
Iser
Wendy B
WB
Hanselman
Keaton B
KB
Wolkow
Catherine A
CA
D000222:Adaptation, Physiological / Q000235:genetics / Q000502:physiology; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D051858:Forkhead Transcription Factors; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008136:Longevity / Q000235:genetics / Q000502:physiology; D009154:Mutation / Q000235:genetics; D018384:Oxidative Stress / Q000502:physiology; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D034622:RNA Interference; D011972:Receptor, Insulin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
D029742:Caenorhabditis elegans Proteins; D051858:Forkhead Transcription Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C108831:daf-16 protein, C elegans; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; C100662:AGE-1 protein, C elegans; C107483:DAF-2 protein, C elegans; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt