The PTEN tumor suppressor homolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway

Abstract

Inactivation of the tumor suppressor PTEN gene is found in a variety of human cancers and in cancer predisposition syndromes. Recently, PTEN protein has been shown to possess phosphatase activity on phosphatidylinositol 3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase. We have identified a homolog of PTEN in Caenorhabditis elegans and have found that it corresponds to the daf-18 gene, which had been defined by a single, phenotypically weak allele, daf-18(e1375). By analyzing an allele, daf-18(nr2037), which bears a deletion of the catalytic portion of CePTEN/DAF-18, we have shown that mutation in daf-18 can completely suppress the dauer-constitutive phenotype caused by inactivation of daf-2 or age-1, which encode an insulin receptor-like molecule and the catalytic subunit of phosphatidylinositol 3-kinase, respectively. In addition, daf-18(nr2037) dramatically shortens lifespan, both in a wild-type background and in a daf-2 mutant background that normally prolongs lifespan. The lifespan in a daf-18(nr2037) mutant can be restored to essentially that of wild type when combined with a daf-2 mutation. Our studies provide genetic evidence that, in C. elegans, the PTEN homolog DAF-18 functions as a negative regulator of the DAF-2 and AGE-1 signaling pathway, consistent with the notion that DAF-18 acts a phosphatidylinositol 3,4,5-trisphosphate phosphatase in vivo. Furthermore, our studies have uncovered a longevity-promoting activity of the PTEN homolog in C. elegans.

Figure 1

CePTEN/daf-18 gene structure. (A) Genomic structure. The exons are depicted as black boxes and numbered. The nr2037 allele contains a deletion that covers part of intron II and exon III. The phosphatase catalytic center, HCKAGKGRTG, abbreviated as the HC motif (double underlined), is deleted in the nr2037 mutant. The ΔBspHI deletion construct, in which exon I and II of the CePTEN/daf-18 gene is removed, is used as a control for the genomic rescue experiment (see Table 1). The 30-bp insertion found in the daf-18(e1375) allele is located in exon IV in the noncatalytic domain of CePTEN/DAF-18. This insertion occurs after codon 574, leading to a frameshift and premature stop codon. (B) Amino acid alignment of the amino-terminal phosphatase domain of CePTEN/DAF-18 and human PTEN. The phosphatase catalytic center, HCKAGKGRTG, is double underlined. Identical residues are shown as white letters in black boxes.

Figure 2

Morphological comparison of daf-18, daf-2, and daf-2; daf-18 strains. (Left) Nomarski micrographs. The head of the animal is shown. The two bulbs of the pharynx are indicated by the arrowheads. (Right) Brightfield micrographs. Transgenic animals, shown in the Bottom two rows, carry an extrachromosomal array of either the functional daf-18 gene (Ex[daf-18(+)]), or a mutant derivative that removes exons I and II (Ex[daf-18(−)]). The animals with genotypes daf-2(e1370) and daf-2(e1370); daf-18(nr2037); Ex[daf-18(+)] are dauer larvae and appear thinner in the brightfield micrographs and have a constricted pharynx as shown in the Nomarski micrographs. Wild-type animals and animals with genotypes daf-18(nr2037), daf-2(e1370); daf-18(nr2037) and daf-2(e1370); daf-18(nr2037); Ex[daf-18(-)] are non-dauer L3 larvae.

Figure 3

Effects of the daf-18(nr2037) mutation on adult lifespans. (A) Lifespans at 25°C. (B) Lifespans at 20°C. The percentage of live animals was plotted as a function of time (days). Wild-type (Bristol N2), daf-18(nr2037), daf-2(e1370), and daf-2(e1370); daf-18(nr2037) strains were compared in parallel. Representatives of two independent sets of experiments are shown. The results were similar in both sets of experiments.

Figure 4

Model for DAF-18 action. AGE-1 is the major signal transducer for the activated DAF-2 insulin receptor-like molecule in regulation of dauer development and lifespan. AGE-1 is predicted to produce PIP3, which negatively regulates lifespan and dauer-formation processes. DAF-18, the PTEN homolog, functions to dephosphorylate PIP3 and thus antagonizes the action of DAF-2 and AGE-1. DAF-18 may also be negatively regulated by DAF-2 (dashed line).