KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain

Abstract

PDZ domain-containing proteins (PDZ proteins) act as scaffolds for protein-protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN-4, a PDZ domain-containing microtubule-associated serine-threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin-4 is required for the long lifespan of daf-2/insulin/IGF-1 receptor mutants. We then show that neurons are crucial tissues for the longevity-promoting role of kin-4. We find that the PDZ domain of KIN-4 binds PTEN, a key factor for the longevity of daf-2 mutants. Moreover, the interaction between KIN-4 and PTEN is essential for the extended lifespan of daf-2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age-related diseases in mammals through their interaction with PTEN.

Keywords: DAF-18/PTEN; KIN-4/MAST kinase; PDZ; aging; insulin/IGF-1 signaling; lifespan.

Figure 1

KIN‐4 is a PDZ protein that is required for the longevity of daf‐2/insulin/IGF‐1 receptor mutants. (a) A plot of percent changes in mean lifespan of rrf‐3(pk1426) [rrf‐3(−)] and rrf‐3(pk1426); daf‐2(e1370) [rrf‐3(−); daf‐2(−)] mutants upon knocking down each candidate PDZ protein‐encoding gene. Orange circles indicate the lifespan results after knocking down each targeted gene. Blue circles indicate the effects of kin‐4 RNAi and gipc‐1/‐2 RNAi on lifespan. A gray circle indicates the lifespan decrease by daf‐16 RNAi that was used as a positive control. Error bars represent standard error of mean (SEM) of two independent lifespan experiments for each RNAi clone. (b, c) Knock‐down of kin‐4 (b) or gipc‐1/‐2 (c) had a larger lifespan‐decreasing effect on rrf‐3(−); daf‐2(−) mutants than on rrf‐3(−) animals. See Supporting information Table S2 for statistics and additional repeats

Figure 2

kin‐4 is required for various phenotypes conferred by reduced insulin/IGF‐1 signaling. (a, b) kin‐4(tm1049) [kin‐4(−)] (a) and kin‐4(nj170) (b) mutation decreased the long lifespan of daf‐2(e1370) [daf‐2(−)] mutants without FUdR treatment. This lifespan‐shortening effect of kin‐4(−) mutation was confirmed by using daf‐2(RNAi) worms with or without FUdR treatment (Supporting information Figure S1d, e). (c) kin‐4(−) fully suppressed the lifespan extension by osm‐5(p813) [osm‐5(−)]. (d) kin‐4(−) mutation did not affect the lifespan increase by eat‐2(ad1116) [eat‐2(−)]. See Supporting information Table S3 for experimental repeats and statistics. (e) kin‐4(−) mutation reduced the dauer formation of daf‐2(−) mutants at 22.5°C (10 independent experiments with ≥33 worms for each trial. Error bars represent SEM. two‐tailed Student's t test. ∗ p < 0.05). See Supporting information Table S4 for statistical analysis. (f) The enhanced oxidative stress resistance of daf‐2(−) mutants was partially suppressed by kin‐4(−) mutation upon treating with 7.5 mM of tert‐Butyl hydroperoxide (t‐BOOH). See Supporting information Table S5 for statistics and experimental repeats

Figure 3

Neurons are crucial tissues for lifespan regulation by KIN‐4. (a) KIN‐4a is predicted to have 4 domains; DUF1908 whose function is not known, pre‐PK that is mainly found in MAST family kinases, protein kinase, and PDZ domains. The deleted part by kin‐4(tm1049) mutation is marked with a black solid line. (b, c) kin‐4::gfp transgene‐encoded protein (KIN‐4::GFP) was mainly expressed in head and tail (c) neurons (arrowheads) and dimly in the intestine (arrow). Scale bar indicates 50 μm. (d) KIN‐4a::GFP increased the shortened lifespan of daf‐2(e1370); kin‐4(tm1049) [daf‐2(−); kin‐4(−)] mutants. (e) kin‐4 transgenic worms did not display longevity. We also found that daf‐2 RNAi did not alter KIN‐4::GFP levels (Supporting information Figure S4d). See Supporting information Table S3 for experimental repeats and statistics. (f) Tissue‐specific promoter‐driven kin‐4a expression [daf‐2(−); kin‐4(−); kin‐4a::gfp] in neurons (daf‐2(e1370); kin‐4(tm1049); rgef‐1p::kin‐4a::gfp) prolonged the shortened lifespan of daf‐2(e1370); kin‐4(tm1049) [daf‐2(−); kin‐4(−)] mutants. (g) Transgenic expression of kin‐4a in the intestine (daf‐2(−); kin‐4(−); ges‐1p::kin‐4a::gfp) did not affect the decreased lifespan of daf‐2(−); kin‐4(−) mutants. (h) The long lifespan of daf‐2(−) mutants was decreased by neuron‐specific (sid‐1(pk3321); uIs69[myo‐2p::mCherry; unc‐119p::sid‐1]) kin‐4 RNAi treatment. (i) Knock‐down of kin‐4 in the intestine (rde‐1(ne213); kbIs7[nhx‐2p::rde‐1; rol‐6(su1006)]) significantly suppressed the long lifespan of daf‐2 mutants. See Supporting information Table S3 for experimental repeats and statistics

Figure 4

DAF‐18/PTEN binds KIN‐4 to affect the longevity of daf‐2 mutants. (a) Percent lifespan changes by knocking down each of 21 genes that encode proteins that bound the PDZ domain of KIN‐4 in rrf‐3(pk1426) [rrf‐3(−)] and rrf‐3(pk1426); daf‐2(e1370) [rrf‐3(−); daf‐2(−)] mutants. Orange circles indicate the effects of RNAi clones on lifespan. Blue circles indicate RNAi clones that displayed bigger lifespan‐decreasing effects on rrf‐3(−); daf‐2(−) mutants than on rrf‐3(−) worms. A red circle indicates an RNAi clone that further increased the long lifespan of rrf‐3(−); daf‐2(−) mutants. Error bars represent SEM of mean survival times from two or three independent RNAi lifespan experiments. (b−d) daf‐18 (b), mel‐11 (c), and mig‐6 (d) RNAi clones had bigger lifespan‐shortening effects on rrf‐3(−); daf‐2(−) mutant than on rrf‐3(−) animals. (e) rps‐0 RNAi treatment further increased the long lifespan induced by daf‐2(−) mutation in an rrf‐3(−) background. See Supporting information Table S7 for experimental repeats and statistical analyses

Figure 5

KIN‐4 physically interacts with DAF‐18/PTEN. (a‐c) Co‐localization of KIN‐4a::GFP and mCherry::DAF‐18. (a) DAF‐18 was mainly expressed in several head neurons (arrowhead). Arrows indicate red fluorescent signals of injection marker. Scale bar is 200 μm. (b) Shown in a blue rectangle is an enlarged picture of the head part in panel A. (c) Deconvolution images of a neuron. Subcellular localization of KIN‐4a::GFP and mCherry::DAF‐18 in a head neuron of mCherry::daf‐18; kin‐4a::gfp; ofm‐1p::rfp animals. Scale bar is 10 μm. (d) The PDZ domain of KIN‐4 (green) overlaps with that of MAST2 (magenta), one of human MAST family kinases. (e) The marked resides (V1186, V1188, I1202, and L1257) are conserved in the predicted structure of the PDZ domain of KIN‐4 (KIN‐4 PDZ, green) with that of human MAST2. F951 is a conserved residue in the C‐terminal region of DAF‐18 (DAF‐18 C, purple, See Supporting information Figure S8). DAF‐18 C from the 951st to the 962nd residues was predicted by substituting each residue of PTEN (2KYL) with a Coot program. (f) HA‐DAF‐18 was co‐immunoprecipitated by immunoprecipitating FLAG‐KIN‐4. IP: immunoprecipitation. IB: immunoblotting (g) A GST pull‐down assay of HA tag‐fused DAF‐18 [HA‐DAF‐18] with GST‐fused KIN‐4 fragment proteins. HA‐DAF‐18 was pulled down by using a PDZ domain‐containing KIN‐4 C‐terminal fragment [C], but not by an N‐terminal KIN‐4 fragment [N] or by a kinase domain‐containing KIN‐4 middle fragment [Mid]. See the upper illustration that depicts the domain regions in KIN‐4. (h) Illustration of wild‐type and deletion mutant DAF‐18 that were used for GST pull‐down assays. Wild‐type DAF‐18 [WT] has the intact DAF‐18 C‐terminal PDZ‐binding motif. DAF‐18 deletion mutant proteins, [Δ4C] and [Δ12C], do not contain the last 4 and 12 amino acids from its C‐terminal ends, respectively. (i) GST pull‐down assays using wild‐type or mutant FLAG‐tagged full‐length DAF‐18 [FLAG‐DAF‐18] with GST‐fused KIN‐4 PDZ domain [GST‐KIN‐4 PDZ] proteins. GST KIN‐4 PDZ strongly bound to FLAG‐DAF‐18 WT [WT] but weakly to FLAG‐DAF‐18 Δ4C [Δ4C] and FLAG‐DAF‐18 Δ12C [Δ12C]

Figure 6

The interaction between KIN‐4 and DAF‐18 is crucial for longevity caused by IIS reduction. (a) Expression pattern of wild‐type [daf‐18 WT], the last four amino acid deletion mutant DAF‐18 transgene [daf‐18 Δ4C] and the last 12 amino acid deletion mutant daf‐18 transgene [daf‐18 Δ12C], fused with mCherry in the posterior intestine cells. (b) daf‐18 WT fully suppressed lifespan reduction by daf‐18(nr2037) [daf‐18(−)] mutation in daf‐2(e1370) [daf‐2(−)] animals. (c) Two independent lines of daf‐18 Δ4C only partially increased the shortened lifespan of daf‐2(−); daf‐18(−) mutants. (d) daf‐18 Δ12C had small lifespan‐increasing effects on the shortened lifespan of daf‐2(−); daf‐18(−) animals. See Supporting information Table S3 for statistics and additional repeats