CAMKII and Calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16
- Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, China
- National Institute of Biological Sciences, Beijing, China
- Institute of Biophysics, Chinese Academy of Sciences, China
Figures
Figure 1
C. elegans Calcineurin TAX-6•CNB-1 directly binds to DAF-16 and negatively regulates DAF-16 nuclear localization.
(A) DAF-16 and TAX-6 form a complex in vivo. Immunoprecipitation of 3xFLAG::DAF-16 expressed under the daf-16 promoter in WT C. elegans pulled down TAX-6::GFP expressed under the tax-6 promoter. The …
Figure 2 with 2 supplements
The effect of tax-6(lf) mutations on DAF-16 localization and lifespan requires unc-43, the CAMKII gene whose effect opposes that of tax-6.
(A) unc-43 RNAi abolished DAF-16 nuclear accumulation in tax-6(ok2065). Adult stage RNAi knockdown of tax-6 extended the WT lifespan (p=0.001) (B) but may have slightly shortened the lifespan of unc-…
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Figure 2—source data 1
The unc-43(null) mutant showed a WT-like lifespan that was epistatic to the longevity effect of tax-6(RNAi), while the unc-43(gf) mutant was long-lived.
(A) The lifespan extension by tax-6 RNAi required unc-43. (B) The longevity of unc-43(n498) was largely dependent on daf-16. (C) The unc-43(n498);cnb-1(ok276) double mutant lived longer than either single mutant.
- https://doi.org/10.7554/eLife.00518.005
Figure 2—figure supplement 1
A screen for the kinase(s) required for the nuclear accumulation of DAF-16::GFP induced by tax-6(null).
https://doi.org/10.7554/eLife.00518.006
Figure 2—figure supplement 2
An unc-43 RNAi clone from the Ahringer library suppressed the nuclear accumulation of DAF-16::GFP induced by tax-6(null).
https://doi.org/10.7554/eLife.00518.007
Figure 3
UNC-43 does not regulate DAF-16 localization through the NSY-1/SEK-1 MAPK kinase pathway.
(A) RNAi knockdown of nsy-1, sek-1, and pmk-2 failed to block the nuclear accumulation of DAF-16::6xHis::GFP in unc-43(gf) mutants. Worms were fed with the indicated RNAi bacteria from hatching and …
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Figure 3—source data 1
km4, the null allele of sek-1, did not shorten the lifespan of unc-43(gf) worms.
- https://doi.org/10.7554/eLife.00518.009
Figure 4
UNC-43 directly binds to and phosphorylates DAF-16.
(A) 3xHA::UNC-43 co-immunoprecipitated with 3xFLAG::DAF-16 and (B) vice versa from lysates of transgenic C. elegans expressing both proteins. The transgenic strains are MQD522 (co-expressing …
Figure 5 with 1 supplement
UNC-43 phosphorylates S286 of DAF-16, and TAX-6•CNB-1 removes this modification.
(A) A mass spectrum (neutral loss-triggered MS3) of a DAF-16 peptide phosphorylated at S286 by UNC-43 in vitro. (B and C) UNC-43 in vitro kinase assays with purified WT or S286A 6xHis-DAF-16 as …
Figure 5—figure supplement 1
Mass spectrum showing a DAF-16 peptide phosphorylated at T240, one of the sites phosphorylated by UNC-43 in vitro.
https://doi.org/10.7554/eLife.00518.012
Figure 6 with 2 supplements
Phosphorylation of DAF-16 at S286 induces the nuclear accumulation of DAF-16 and extends lifespan.
The S286A mutation prevented DAF-16::6xHis::GFP from accumulating in the nucleus in unc-43(n498gf) (A) or tax-6(ok2065) animals (B). Unlike the DAF-16::6xHis::GFP transgene, DAF-16(S286A)::6xHis::GFP…
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Figure 6—source data 1
The DAF-16(S286A) mutation largely suppressed the longevity induced by either unc-43(gf) or tax-6(null), while the DAF-16(S286D) mutation extended lifespan.
(A) Mutation of DAF-16 S286 to A partially suppressed the unc-43(gf) induced longevity. (B) Mutation of DAF-16 S286 to A partially suppressed the longevity of tax-6(null). (C) daf-16(mu86) animals expressing DAF-16 (S286D)::GFP had a longevity phenotype.
- https://doi.org/10.7554/eLife.00518.014
Figure 6—figure supplement 1
UNC-43 does not phosphorylate T240 in vivo to promote DAF-16 nuclear accumulation.
(A) DAF-16(T240A)::6xHis::GFP accumulated in the intestinal nuclei even in the absence unc-43(gf). This is incompatible with T240 being the UNC-43 phosphorylation site because mutating such a site …
Figure 6—figure supplement 2
Similar expression levels of DAF-16(S286D)::6xHis::GFP and DAF-16::6xHis::GFP in three strains used in Figure 6F.
GFP fusion proteins in whole-worm lysates were quantified by anti-GFP WB. SDS-PAGE and anti-tubulin WB show equal loading of total proteins. For hqEx168 and hqEx174, because the transgene arrays …
Figure 7 with 2 supplements
The longevity of daf-2 mutants was partially suppressed by unc-43(null) and further enhanced by unc-43(gf) or tax-6(null).
unc-43(n498), a gain-of-function allele (A), and tax-6(ok2065), a null allele (B), each extended lifespan in the WT background and further enhanced the longevity of daf-2(RNAi) animals. (C) The daf-2…
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Figure 7—source data 1
The long lifespan of daf-2(RNAi) animals was further extended by either unc-43(gf) or tax-6(null), but shortened by unc-43(null).
(A) Both unc-43(gf) and tax-6(null) further increased the lifespan of daf-2 RNAi animals. (B) unc-43 was partially required for the long lifespan of daf-2 animals. (C) The DAF-16 S286A mutation appears not to affect the daf-2 longevity.
- https://doi.org/10.7554/eLife.00518.018
Figure 7—figure supplement 1
The DAF-16 S286A mutation appears not to affect daf-2 longevity.
(A)–(B) Two repeat experiments showing similar lifespan extension by daf-2 RNAi of daf-16(mu86) worms expressing DAF-16::6xHis::GFP (hqEx192 and hqEx193) or DAF-16(S286A)::6xHis::GFP (hqEx365 and hqE…
Figure 7—figure supplement 2
In the unc-43(n498gf) mutant, in which DAF-16 accumulates in the nucleus, the amount of DAF-16/14-3-3 complex is reduced.
DAF-16::6xHis::GFP was immunoprecipitated from worms carrying a WT or the gf allele of unc-43. The amount of 14-3-3 present in the lysate and that associated with DAF-16::6xHis::GFP were visualized …
Figure 8
Insulin signaling overpowers CAMKII and Calcineurin in regulation of DAF-16 localization in C. elegans.
(A) tax-6(gf) or unc-43(null) failed to abolish DAF-16 nuclear accumulation induced by daf-2(RNAi). (B) tax-6(null) or unc-43(gf) failed to overcome the inhibition of DAF-16 nuclear localization by d…
Figure 9 with 3 supplements
CAMKII and Calcineurin regulate phosphorylation of mouse FOXO3 at S298.
(A) Sequence alignment of selected FOXO homologs from nematode species to human. The region containing DAF-16 T240 and S286 is shown. T240 is conserved in all FOXO homologs. S286 is less conserved, …
Figure 9—figure supplement 1
Analysis of FOXO3 phosphorylation sites in vivo.
Mass spectra showing two phosphorylation sites S252 (A) and S279 (B) identified from FLAG-mFOXO3 expressed in HEK293T cells. Mass spectrum identifying a third phosphorylation site S298 is shown in …
Figure 9—figure supplement 2
CAMIIA had no effect on S253 phosphorylation of human FOXO3 (corresponding to S252 of mouse FOXO3) in vivo.
(A) Transfection of CAMKIIA into HEK293T cells failed to increase phosphorylation of hFOXO3 at S253. (B) Overexpression of Calcineurin A into HEK293T cells did not reduce phosphorylation of human …
Figure 9—figure supplement 3
In vitro Calcineurin phosphatase assay on FOXO3.
(A) The S298-containing P3 fragment of mouse FOXO3 was first phosphorylated in vitro by CAMKIIA in the presence of [32P]-γ-ATP, then incubated with Calcineurin after the in vitro kinase reaction. …
Figure 10
CAMKII and Calcineurin regulate the transcriptional activity of FOXO3.
(A)–(D) HEK293T cells were transfected with the indicated constructs together with a 3xIRS-firefly luciferase reporter and a TK-renilla luciferase reporter. Mean ± SD of firefly/renilla luciferase …
Additional files
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Supplementary file 1
(A) C. elegans strains used. (B) Oligos for genotyping.
- https://doi.org/10.7554/eLife.00518.027
