PDZ domain-containing proteins (PDZ proteins) comprise a large family of scaffold proteins that participate in organizing components in various signal transduction pathways. However, it remains largely unknown regarding the role of PDZ proteins in C. elegans insulin/IGF-1 signaling (IIS) pathway, which regulates lifespan, stress resistance and dauer formation. To address this issue, we first performed an RNAi-based lifespan screen targeting 49 out of 70 genes that encode putative PDZ proteins using wild-type (N2) and long-lived
daf-2/insulin/IGF-1 receptor mutant animals. We found that knockdown of
kin-4, a PDZ-containing microtubule-associated serine-threonine (MAST) kinase, largely suppressed the long lifespan of
daf-2(
e1370) mutants. We confirmed this lifespan result by using
kin-4(
tm1049) deletion mutants.
kin-4(
tm1049) mutations also significantly decreased the enhanced oxidative stress resistance caused by
daf-2(
e1370) mutations. We then showed that overexpression of
kin-4 fused with GFP, which was detected mainly in the pharynx and the intestine, was able to increase lifespan. These data suggest that
kin-4 is necessary and sufficient for longevity. Next, we performed an extensive yeast two-hybrid screen to identify proteins that interacted with the PDZ domain of KIN-4. Among the 41 candidates, we identified DAF-18/PTEN phosphatase, which acts downstream of DAF-2 and is required for the longevity of
daf-2 mutants. We confirmed the physical interaction between KIN-4 and DAF-18 by using in vitro pull down and mammalian cell culture-based co-immunoprecipitation assays. We showed that the PDZ domain of KIN-4 bound to a canonical PDZ-binding motif located at the C-terminal end of DAF-18. Importantly, disruption of the interaction between KIN-4 and DAF-18 partially but significantly suppressed the long lifespan of
daf-2(
e1370) mutants. Overall, our data suggest that KIN-4 contributes to longevity conferred by reduced IIS via binding DAF-18 through its PDZ domain. We are currently examining which longevity transcription factors among DAF-16/FOXO, HSF-1/heat shock factor 1 and SKN-1/NRF2 mediate the longevity signals transduced from the interaction between KIN-4 and DAF-18. Our study will help understand how PDZ scaffold proteins regulate organismal lifespan by participating in IIS pathway.