Giving DAF-16 the upper hand: a transcriptional switch in the IGF pathway?

The insulin-like growth factor (IGF) pathway is one of the longest-known and well-studied regulators of longevity. Extracellular signals (insulin-like peptides) activate insulin-receptor homologs (in worm, DAF-2) which in turn recruit and activate phosphoinositol 3-kinases (AGE-1). PI3Ks convert PIP2 into PIP3, which tethers and recruits other kinases such as AKT-1. Eventually, activation of these upstream kinases results in phosphorylation and inactivation of the longevity assurance gene DAF-16, which encodes a transcription factor that activates (among many other things) stress resistance genes.

To recap: High DAF-2 and AGE-1 activity => low DAF-16 activity => shorter lifespan. Lower DAF-2 or AGE-1 => high DAF-16 activity => longer lifespan. The lifespan extension of daf-2 or age-1 mutants absolutely requires wildtype DAF-16.

From this simple model, it would seem that the levels of DAF-2 agonists would run the show; DAF-16 activity would simply be a readout of signaling upstream of the insulin-like growth factor receptor. As is so often the case, however, this simple model turns out to be simplistic: DAF-16 plays an active role in determining the signaling through this pathway, as revealed by Tazearslan et al.:

Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance

Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP2 to form PIP3, a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust—implying that maternally contributed trace amounts of PI3K activity or of PIP3 block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP3-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO–dependent and –independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode.

PIP3 is necessary for the membrane tethering and activation of a great many kinases; thus, profound defects in PI3K activity would be predicted to result in profound defects in phosphoprotein signaling. Indeed, the authors see dramatic effects on in vitro kinase activity and steady-state phosphoprotein levels in their age-1 mutants — not just for known downstream targets of the IGF pathway but for bulk protein.

Note the importance of using F2 age-1 mutants, i.e., animals that are themselves the offspring of homozygous age-1 mutants: Wildtype AGE-1 activity in the parent, even from a single copy of the gene, is sufficient to maternally rescue PI3K activity in the offspring to some extent. This results in a weaker phenotype in the F1s than in their progeny, the F2s, who completely lack PI3K activity and consequently enjoy much longer lifespan and higher resistance to stress. There’s no way to find out about F3s, since the F2s are completely sterile.

In the absence of upstream IGF signaling, downstream effector kinases would not be activated by phosphorylation. Here’s where the story throws us a curve-ball: As predicted, in the profoundly long-lived age-1 F2’s, the effector kinases are inactive — but the transcripts encoding them are also downregulated. In the absence of upstream signaling, there’s no longer a kinase cascade bearing down on DAF-16, which therefore remains unphosphorylated and active. And what does DAF-16 do? It heads to the nucleus and transcriptionally silences the genes encoding the upstream kinases DAF-2, AGE-1 and others — in other words, DAF-16 turns off the genes that could turn off DAF-16.

It’s a feedback loop! Disinhibition of DAF-16 by lowering PIP3 levels is self-sustaining, because disinhibited DAF-16 lowers transcription of PI3Ks, thereby further lowering PIP3 levels. The authors argue that this arrangement represents a biological switch between a short-lived “reproductive state” and a non-reproducing “longevity state”, characterized by DAF-16 activation of stress-resistance and other types of longevity assurance genes.

They have a point, but I think they might be overstating the “switchiness” of this switch. One of the main features of a switch (of the sort that motivates the analogy) is that it can be on or off but not halfway between the two — there are disequilibrating forces at work that push it away from the middle and toward either pole. Unfortunately for the authors’ interpretation, most of the work done in this field to date has been done in the equivalent of the F1 age-1 mutants, where a combination of partial gene function and maternal factors have placed worms somewhere between “all reproduction” and “all longevity”. The existence of these animals does somewhat mitigate the argument that this system represents a binary switch in the strictest sense of the word.

To be fair to the authors, they acknowledge this, most clearly in Figure 6, a model that allows for three states — reproductive (low DAF-16, e.g., wildtype), longevity (high DAF-16, e.g. daf-2 or age-1 F1), and “hyperlongevity” (extremely high DAF-16 that completely shuts off PI3K activity, e.g. age-1 F2). The distinction between the two long-lived cases is somewhat elided in the Discussion, where the authors emphasize the feedback loop and consider the longevity states as though they were the same.

ResearchBlogging.orgTazearslan, C., Ayyadevara, S., Bharill, P., & Shmookler Reis, R. (2009). Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance PLoS Genetics, 5 (4) DOI: 10.1371/journal.pgen.1000452