CAMKII and Calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16
Multiple signaling pathways , including insulin/IGF-1 signaling ( IIS ) , germline signaling , mitochondrial signaling , and the signaling pathways induced by dietary restriction , regulate aging in C . elegans ( Kenyon , 2005; Greer and Brunet , 2008 ) . The best characterized is the IIS pathway , which includes the upstream insulin/IGF-1 receptor DAF-2 and the downstream FOXO transcription factor DAF-16 . Signals from DAF-2 are transmitted through AGE-1 ( phosphoinositide 3-kinase ) and PDK-1 ( phosphoinositol-dependent kinase-1 ) to AKT-1 , AKT-2 , and SGK-1 , which phosphorylate DAF-16 and prevent it from translocating to the nucleus to activate a pro-longevity gene network . Reduction-of-function mutations of the kinase genes in the IIS pathway , from daf-2 to akt-1 , akt-2 , and sgk-1 , all extend lifespan in a daf-16-dependent manner ( Kenyon et al . , 1993; Morris et al . , 1996; Kimura et al . , 1997; Hertweck et al . , 2004 ) . Lifespan extension by reduced IIS is also observed in other species , including flies and mice , indicating that the pathway is conserved evolutionarily ( Kenyon , 2005; Greer and Brunet , 2008 ) . Compared with the kinases in the C . elegans IIS pathway , little is known about the protein phosphatases that neutralize the effects of the kinases . The only known example is PPTR-1 , a B56 regulatory subunit of PP2A , which directs PP2A to dephosphorylate AKT-1 at T350 , thereby inactivating the kinase ( Padmanabhan et al . , 2009 ) . DAF-18 , the C . elegans PTEN , is a phosphatidylinositol 3 , 4 , 5-trisphosphate ( PIP3 ) 3-phosphatase ( Ogg and Ruvkun , 1998 ) . The phosphatase for DAF-2 has not been identified , nor the one that regulates DAF-16 . Previously , loss-of-function ( lf ) mutants for tax-6 and cnb-1 , which encode the catalytic and regulatory subunits of C . elegans Calcineurin , respectively , were found to live longer than wild-type ( WT ) worms ( Dong et al . , 2007 ) . In mammalian systems , Calcineurin ( PP2B ) is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase that has diverse functions and affects both T cell activation and heart development ( Crabtree , 1999 ) . In C . elegans , Calcineurin regulates body size , thermotaxis , muscle contraction , and lifespan ( Bandyopadhyay et al . , 2002; Kuhara et al . , 2002; Lee et al . , 2004; Dong et al . , 2007 ) . The longevity phenotype of tax-6 ( lf ) is partially dependent on daf-16 ( Dong et al . , 2007 ) . More recent studies have shown that C . elegans Calcineurin can regulate lifespan by suppressing autophagy ( Dwivedi et al . , 2009 ) or inactivating CRTC-1 , a co-activator of CREB ( Mair et al . , 2011 ) . However , direct targets of worm Calcineurin have not been identified . In the current work , we addressed how worm Calcineurin TAX-6•CNB-1 regulates lifespan . We discovered that DAF-16 was phosphorylated and activated by UNC-43 at the serine 286 ( S286 ) site . The phosphoryl group was removed by TAX-6•CNB-1 . UNC-43 and TAX-6•CNB-1 therefore regulate C . elegans lifespan through the reversible phosphorylation of DAF-16 . This regulatory mechanism has a different mode of action from the canonical IIS pathway because the phosphorylation activates , rather than represses , DAF-16 . Activation of DAF-16 by UNC-43 occurs in response to different types of stress signals , such as heat , starvation , and oxidation . UNC-43 and TAX-6•CNB-1 can regulate DAF-16 independently of IIS , and the two signaling mechanisms appear to crosstalk , leading to coordinated action on DAF-16 . We also show that the regulation of FOXO by CAMKII and Calcineurin is conserved in mammalian cells .
A genetic analysis has shown that a daf-16 ( null ) allele partially suppresses the longevity of the tax-6 ( lf ) mutant ( Dong et al . , 2007 ) . This observation suggested that daf-16 is either a direct or indirect downstream target of C . elegans Calcineurin; alternatively , it acts independently . To sort through these possibilities , we immunoprecipitated the 3xFLAG::DAF-16 protein using an anti-FLAG antibody from the lysate of MQD82 , a transgenic worm strain that expresses this fusion protein and TAX-6::GFP ( Figure 1A , lane 1 ) . TAX-6::GFP from this lysate was co-precipitated with the FLAG antibody ( Figure 1A , lane 4 ) , but no TAX-6::GFP was co-precipitated from the lysate of MQD2 , a strain expressing only TAX-6::GFP ( Figure 1A , lanes 2 and 5 ) . From the mixed lysates of two other transgenic strains , one expressing 3xFLAG::DAF-16 and the other expressing GFP , the GFP protein also failed to be precipitated by the FLAG antibody ( Figure 1A , lanes 3 and 6 ) . This result suggested that TAX-6 and DAF-16 interacted with each other in vivo . To determine whether the interaction was direct , we individually purified the recombinant His-tagged DAF-16 and C . elegans Calcineurin in the form of TAX-6•GST-CNB-1 and then mixed them together before pulling down DAF-16 with nickel beads . Indeed , TAX-6•GST-CNB-1 was pulled down successfully using His-tagged DAF-16 ( Figure 1B ) . Further analysis suggested that the C-terminal region of DAF-16 mediates the interaction with Calcineurin ( Figure 1C ) . Together , these results raised the interesting possibility that Calcineurin might directly regulate DAF-16 in C . elegans . 10 . 7554/eLife . 00518 . 003Figure 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 lysates were obtained from transgenic strains MQD82 ( co-expressing 3xFLAG::DAF-16 and TAX-6::GFP ) , MQD2 ( expressing TAX-6::GFP ) , MQD89 ( expressing 3xFLAG::DAF-16 ) , and CF1553 ( expressing GFP under a sod-3 promoter ) . ( B ) Calcineurin directly binds to DAF-16 . Purified recombinant TAX-6•GST-CNB-1 was pulled down with Ni-NTA beads through its interaction with purified His-tagged DAF-16 . ( C ) The C-terminal region of DAF-16 most likely mediates the interaction with Calcineurin . GST-DAF-16 ( F-C ) , but not GST , GST-DAF-16 ( N ) or GST-DAF-16 ( N-F ) , pulled down TAX-6::GFP expressed in C . elegans ( strain MQD5 ) . The DAF-16 C-terminal region alone was not stable . Asterisk indicates full-length GST or GST fusion proteins . ( D ) DAF-16::6xHis::GFP is diffusely distributed in the WT animals but concentrated in the nucleus in tax-6 ( ok2065 ) animals . All GFP images shown in this paper are of L4 animals at 20°C unless otherwise indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 003 In WT animals , DAF-16 is phosphorylated by AKT and diffusely distributed throughout the cell , whereas in long-lived IIS mutants , transcriptionally active DAF-16 accumulates in the nucleus ( Henderson and Johnson , 2001; Lin et al . , 2001 ) . To determine whether DAF-16 translocates to the nucleus in long-lived Calcineurin mutants , we expressed DAF-16::6xHis::GFP using a daf-16 promoter in the tax-6 ( ok2065 ) background . The functionality of this transgene was verified in daf-2 ( e1370ts ) ;daf-16 ( mu86 ) animals; the double mutant expressing this transgene displayed the dauer formation-constitutive ( Daf-c ) and longevity phenotypes of the daf-2 ( e1370ts ) single mutant ( not shown ) . DAF-16::6xHis::GFP clearly accumulated in the nuclei of different types of cells in tax-6 ( ok2065 ) ( Figure 1D ) , similar to daf-2 ( lf ) and akt-1;akt-2 ( RNAi ) mutants ( Henderson and Johnson , 2001 ) . The above results strongly suggested that Calcineurin directly inhibited DAF-16 , presumably by dephosphorylating it . This result is in contrast to the transmission of IIS signaling to DAF-16 , during which phosphorylation of DAF-16 by AKT inhibits its nuclear accumulation . Thus , AKT and Calcineurin regulate the phosphorylation of different sites on DAF-16; otherwise , tax-6 ( lf ) would have a lifespan phenotype opposite to that of akt-1;akt-2 ( lf ) or daf-2 ( lf ) . In contrast , they all live longer than WT worms . Therefore , Calcineurin must counteract a different kinase that activates DAF-16 . We reasoned that nuclear accumulation of DAF-16 in the tax-6 ( null ) background should be dependent on a kinase that initiated the phosphorylation and nuclear translocation of DAF-16 . Inactivation of this kinase should abolish the nuclear accumulation of DAF-16::GFP in tax-6 ( null ) animals . We thus conducted an RNAi screen of kinase genes using the DAF-16::6xHis::GFP reporter in tax-6 ( ok2065 ) worms , in which the GFP signal accumulates in the nucleus ( Figure 2—figure supplement 1 ) . We obtained from the Ahringer library a strong suppressor of nuclear DAF-16::GFP in an RNAi clone that targets unc-43 ( Figure 2—figure supplement 2 ) . The suppression was confirmed by an independent , homemade unc-43 RNAi construct ( Figure 2A ) . The unc-43 gene encodes the only Ca2+/calmodulin-dependent serine/threonine protein kinase , type II ( CAMKII ) , in C . elegans ( Reiner et al . , 1999 ) . UNC-43 and Calcineurin have opposing functions in locomotion and egg laying , two behavioral phenotypes that are regulated by G protein signaling ( Reiner et al . , 1999; Bandyopadhyay et al . , 2002 ) . However , they also have non-overlapping functions because unc-43 ( lf ) ;cnb-1 ( null ) double mutant animals arrest at the L1 larval stage , whereas both single mutants are viable ( Reiner et al . , 1999; Bandyopadhyay et al . , 2002 ) . 10 . 7554/eLife . 00518 . 004Figure 2 . 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-43 ( n498n1186 ) , a putative null mutant ( p=0 . 04 ) ( C ) . A constitutively active gain-of-function mutation , unc-43 ( n498 ) , caused DAF-16::6xHis::GFP to accumulate in the nucleus ( D ) and extended lifespan in a largely daf-16-dependent manner ( E ) . p<0 . 001 for daf-16 ( mu86 ) ;unc-43 ( gf ) vs daf-16 ( mu86 ) or unc-43 ( gf ) . The log-rank p values are reported for all lifespan data in this study . ( F ) The unc-43 ( gf ) ;cnb-1 ( null ) double mutant has a longer lifespan than both the unc-43 ( gf ) and cnb-1 ( null ) mutants , and all three mutant strains live longer than WT animals . p<0 . 001 for WT vs any mutant , and p<0 . 001 for unc-43 ( gf ) ;cnb-1 ( null ) vs unc-43 ( gf ) and cnb-1 ( null ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 00410 . 7554/eLife . 00518 . 005Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 00510 . 7554/eLife . 00518 . 006Figure 2—figure supplement 1 . A screen for the kinase ( s ) required for the nuclear accumulation of DAF-16::GFP induced by tax-6 ( null ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 00610 . 7554/eLife . 00518 . 007Figure 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 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 007 To verify that UNC-43 and Calcineurin regulate DAF-16 antagonistically , we examined whether unc-43 ( RNAi ) could suppress the longevity of Calcineurin loss-of-function mutants and whether unc-43 ( gf ) had the same phenotype as tax-6 ( lf ) . Although RNAi knockdown of tax-6 during adulthood increased the lifespan of WT worms ( Figure 2B , Figure 2—source data 1A ) , it failed to increase the lifespan of unc-43 ( null ) animals ( Figure 2C , Figure 2—source data 1A ) . This observation is consistent with the hypothesis that Calcineurin removes the activating phosphorylation on DAF-16 caused by UNC-43 . We then examined n498 , an unc-43 gain-of-function ( gf ) allele harboring a missense mutation that results in constitutive activation of the kinase ( Park and Horvitz , 1986; Reiner et al . , 1999 ) . This unc-43 ( gf ) allele caused DAF-16::6xHis::GFP to accumulate in the nucleus ( Figure 2D ) , phenocopying tax-6 ( lf ) ( Figure 2A ) . Furthermore , the unc-43 ( n498 ) mutant lived 80% longer than the WT animals , and this lifespan extension was largely dependent on daf-16 ( Figure 2E , Figure 2—source data 1B ) . Consistent with UNC-43 activating DAF-16 to prolong lifespan and TAX-6•CNB-1 antagonizing such action , we found that the unc-43 ( gf ) ;cnb-1 ( lf ) double mutant lived even longer than either single mutant ( Figure 2F , Figure 2—source data 1C ) . Previous studies have shown that UNC-43 activates NSY-1 and SEK-1 , two kinases within the MAP kinase pathway , to repress str-2 expression ( Sagasti et al . , 2001 ) . To determine whether UNC-43 activated DAF-16 through NSY-1 ( MAPKKK ) and SEK-1 ( MAPKK ) , we treated unc-43 ( n498 ) worms expressing the DAF-16::6xHis::GFP transgene with nsy-1 and sek-1 RNAi and found that neither gene affected DAF-16 nuclear accumulation induced by unc-43 ( gf ) ( Figure 3A ) . RNAi against pmk-2 , which encodes a p38 MAP kinase ( Kim et al . , 2002; Tanaka-Hino et al . , 2002 ) , also had no effect ( Figure 3A ) . sek-1 regulates DAF-16 localization in response to oxidative stress ( Kondo et al . , 2005 ) . To ascertain the role of sek-1 in the nuclear accumulation of DAF-16 induced by unc-43 ( gf ) , we crossed the DAF-16::6xHis::GFP animal to a unc-43 ( gf ) ;sek-1 ( null ) double mutant . Similar to the RNAi treatment , the km4 ( null ) allele of sek-1 failed to prevent DAF-16 nuclear localization in unc-43 ( gf ) animals ( Figure 3B ) . Moreover , the unc-43 ( gf ) ;sek-1 ( null ) double mutant did not live a shorter life than unc-43 ( gf ) animal ( Figure 3C , Figure 3—source data 1 ) . Thus , we conclude that UNC-43 does not activate DAF-16 through the NSY-1/SEK-1 MAP kinase pathway . 10 . 7554/eLife . 00518 . 008Figure 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 imaged at the L4 stage . ( B ) Similar to sek-1 RNAi , the sek-1 ( km4 ) mutation did not eliminate DAF-16 nuclear localization in unc-43 ( gf ) worms . ( C ) km4 , a null allele of sek-1 , did not shorten the lifespan of WT or unc-43 ( gf ) animals . In contrast , km4 may have slightly extended their lifespan . p=0 . 001 for sek-1 ( null ) vs WT and p=0 . 047 for unc-43 ( gf ) ; sek-1 ( null ) vs unc-43 ( gf ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 00810 . 7554/eLife . 00518 . 009Figure 3—source data 1 . km4 , the null allele of sek-1 , did not shorten the lifespan of unc-43 ( gf ) worms . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 009 To test whether DAF-16 is a direct substrate of UNC-43 , we constructed transgenic strains that co-expressed 3xHA::UNC-43 and 3xFLAG::DAF-16 ( MQD522 ) or expressed either 3xHA::UNC-43 ( MQD530 ) or 3xFLAG::DAF-16 ( MQD89 ) under the control of own promoter . The 3xHA::UNC-43 protein was found in the immunoprecipitate of 3xFLAG::DAF-16 and vice versa ( Figure 4A–B ) , suggesting that UNC-43 and DAF-16 can form a complex in vivo . Using purified recombinant proteins , we found that GST-UNC-43 but not GST or calmodulin ( CAM ) , was pulled down by nickel beads via 6xHis-DAF-16 ( Figure 4C ) . 10 . 7554/eLife . 00518 . 010Figure 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 3xHA::UNC-43 and 3xFLAG::DAF-16 ) , MQD530 ( expressing 3xHA::UNC-43 ) , and MQD89 ( expressing 3xFLAG::DAF-16 ) . ( C ) UNC-43 can directly bind to DAF-16 . Purified GST-UNC-43 but not CAM or the GST control was pulled down by Ni-NTA beads through its interaction with 6xHis-DAF-16 . A Coomassie gel is shown at the bottom . ( D ) – ( E ) In vitro kinase assays in the presence of [32P]-γ-ATP , in which purified GST-UNC-43 directly phosphorylated His-tagged DAF-16 ( D ) or DAF-16 ( N-F ) fragments ( E ) . The Coomassie-stained gel is shown below the autoradiograph . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 010 We then asked whether UNC-43 phosphorylates DAF-16 . We performed an in vitro kinase assay using purified GST-UNC-43 , Ca2+/CAM , and 6xHis-DAF-16 in the presence of [32P]-γ-ATP . GST-UNC-43 readily phosphorylated DAF-16 in vitro ( Figure 4D ) . To map the phosphorylation site ( s ) , truncated DAF-16 proteins were used as kinase substrates . UNC-43 phosphorylated the DAF-16 N-F fragment ( 1–267 aa ) but not the N-terminal fragment ( 1–142 aa ) ( Figure 4E ) , suggesting that the phosphorylation site ( s ) resides in the region containing the forkhead domain ( 143–267 aa ) and/or the C-terminal region ( 268–508 aa ) . A mass spectrometry ( MS ) analysis of the full-length 6xHis-DAF-16 after the in vitro kinase reaction identified two DAF-16 residues T240 and S286 that were phosphorylated by UNC-43 ( Figure 5—figure supplement 1 , Figure 5A ) . T240 and S286 are among the predicted CAMKII sites ( RXXS/T or S/TXD ) . To confirm the MS result , we made two antibodies , one that specifically recognized phospho-T240 and one that specifically recognized phospho-S286 . Using these antibodies , we verified that DAF-16 was indeed phosphorylated by UNC-43 at T240 and S286 in vitro ( Figure 5B–C ) . 10 . 7554/eLife . 00518 . 011Figure 5 . 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 substrate . DAF-16 phosphorylation was detected with antibodies specific for either phospho-T240 ( B ) or phospho-S286 ( C ) . ( D and E ) UNC-43 phosphorylates DAF-16 at S286 but not T240 in vivo . Phosphorylated DAF-16::6xHis::GFP was immunoprecipitated from unc-43 ( wt ) or unc-43 ( gf ) animals using antibodies specific for either phospho-S286 ( D ) or phospho-T240 ( E ) and visualized by blotting with an anti-GFP antibody . Transgenic strains expressing S286A or T240A DAF-16::6xHis::GFP served as negative controls . All strains carry the daf-16 null allele mu86 in the background . ( F ) TAX-6•CNB-1 dephosphorylates DAF-16 specifically at S286 . Purified 6xHis-DAF-16 was phosphorylated by UNC-43 in vitro and then incubated with purified TAX-6•GST-CNB-1 after heat inactivation of UNC-43 . Phospho-T240 , phospho-S286 , and total DAF-16 levels were assayed by western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01110 . 7554/eLife . 00518 . 012Figure 5—figure supplement 1 . Mass spectrum showing a DAF-16 peptide phosphorylated at T240 , one of the sites phosphorylated by UNC-43 in vitro . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 012 To confirm whether DAF-16 is phosphorylated in vivo at both sites in an UNC-43-dependent manner , we immunoprecipitated phospho-DAF-16::6xHis::GFP from daf-16 ( null ) ;unc-43 ( gf ) or daf-16 ( null ) mutants using the phospho-DAF-16-specific antibodies and followed with anti-GFP immunoblotting . Interestingly , there was a marked increase of DAF-16 S286 phosphorylation , but not T240 phosphorylation , in the unc-43 ( gf ) mutant ( Figure 5D–E ) . S286A and T240A mutations completely abolished phosphorylation on the respective sites ( Figure 5C–E ) . We thus concluded that UNC-43 phosphorylates DAF-16 specifically on S286 . Apparently , T240 phosphorylation by UNC-43 does not occur in vivo under the tested condition . To test whether TAX-6 dephosphorylates DAF-16 at S286 , we phosphorylated recombinant DAF-16 in vitro with UNC-43 and then incubated it with purified TAX-6•GST-CNB-1 . As shown in Figure 5F , phosphorylation of DAF-16 at S286 was dramatically reduced by TAX-6•GST-CNB-1 , while phosphorylation of T240 remained unchanged . To validate the role of S286 in UNC-43- and TAX-6-regulated DAF-16 localization and longevity , we introduced the DAF-16 ( S286A ) ::6xHis::GFP transgene into daf-16 ( mu86 ) ;unc-43 ( gf ) and daf-16 ( mu86 ) ;tax-6 ( lf ) mutants . While DAF-16::6xHis::GFP accumulated in the nuclei of tax-6 ( lf ) and unc-43 ( gf ) mutants , DAF-16 ( S286A ) ::6xHis::GFP distributed diffusely throughout the cell in the same mutant backgrounds ( Figure 6A–B ) , and the pattern resembled that of DAF-16::6xHis::GFP in WT worms ( Figure 1D ) . In C . elegans whose WT copy of DAF-16 was replaced by DAF-16 ( S286A ) , the lifespan extension by either unc-43 ( gf ) or tax-6 ( lf ) was greatly suppressed ( Figure 6C–D , Figure 6—source data 1A , B ) . Thus , the S286 residue is required for DAF-16-dependent effects of unc-43 ( gf ) and tax-6 ( lf ) , verifying that S286 is the regulatory site . In comparison , the constitutive nuclear localization phenotype of DAF-16 ( T240A ) contradicts with the idea that UNC-43 phosphorylates T240 to promote DAF-16 nuclear accumulation ( Figure 6—figure supplement 1 ) . Rather , it corroborates the observation that UNC-43 does not phosphorylate T240 in vivo ( Figure 5E ) . 10 . 7554/eLife . 00518 . 013Figure 6 . 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 failed to restore the long lifespan in daf-16 ( mu86 ) ;unc-43 ( n498gf ) ( C ) or daf-16 ( mu86 ) ;tax-6 ( ok2065 ) mutants ( D ) . p<0 . 001 for unc-43 ( gf ) vs daf-16;unc-43 ( gf ) ;hqEx182 in ( C ) and tax-6 ( null ) vs . daf-16;tax-6 ( null ) ;hqEx221 in ( D ) . The S286D mutation caused DAF-16::6xHis::GFP to accumulate in the nucleus ( E ) and extended lifespan in the daf-16 ( mu86 ) background ( F ) . p<0 . 001 , daf-16;hqEx174/168 vs . daf-16;hqIs9 in ( F ) . 2 hr of heat stress at 28°C , 5 min of paraquat treatment , or 20 hr of food deprivation induced nuclear accumulation of DAF-16::6xHis::GFP in the WT animals but not the unc-43 ( n498n1186 ) mutants ( G ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01310 . 7554/eLife . 00518 . 014Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01410 . 7554/eLife . 00518 . 015Figure 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 to alanine was expected to inhibit DAF-16 nuclear accumulation . Rather , this result confirms that T240 is an AKT phosphorylation site ( Lin et al . , 2001 ) . ( B ) 80% of the worms expressing a GFP-tagged DAF-16 ( T240A-S286A ) mutant protein under a daf-16 promoter in the unc-43 ( n498 ) ;daf-16 ( mu86 ) background arrested as dauers , and the GFP signal was predominantly in the head neurons . A few individuals escaped dauer arrest and grew into adults . These animals displayed a strong nuclear accumulation of DAF-16 ( T240A-S286A ) in body-wall muscles and hypodermis but a weakened nuclear accumulation in the intestine compared to those expressing WT DAF-16 . In comparison , the S286A mutation prevented DAF-16 from accumulating in the nucleus when expressed in the same background ( Figure 6A ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01510 . 7554/eLife . 00518 . 016Figure 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 were not chromosomally integrated , GFP positive worms were hand-picked under the microscope and pooled to make lysates . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 016 To mimic phosphorylation by UNC-43 , we mutated DAF-16 S286 to aspartic acid . When expressed in daf-16 ( mu86 ) animals , the DAF-16 ( S286D ) ::6xHis::GFP protein accumulated in the nucleus , whereas the WT DAF-16::6xHis::GFP protein was cytoplasmic , as expected ( Figure 6E ) . Moreover , the phosphomimetic of DAF-16 , but not the WT version , significantly increased the C . elegans lifespan in daf-16 ( null ) animals ( Figure 6F , Figure 6—source data 1C ) . The effect was not due to higher protein amounts of DAF-16 ( S286D ) because DAF-16 ( S286D ) ::6xHis::GFP and DAF-16::6xHis::GFP were expressed at similar levels in these transgenic animals ( Figure 6E , Figure 6—figure supplement 2 ) . Taken together , the S286D mutation mimicked the effect of unc-43 ( gf ) and tax-6 ( lf ) on DAF-16 , whereas the S286A mutation of DAF-16 blocked the effects of unc-43 ( gf ) and tax-6 ( lf ) . Thus , phosphorylation of S286 is critical for CAMKII and Calcineurin to regulate DAF-16 localization and longevity . S286 is different from the known AKT phosphorylation sites ( T54 , S238 , T240 , and S312 for DAF-16b ) of DAF-16 ( Lin et al . , 2001 ) . Therefore , UNC-43 and TAX-6•CNB-1 most likely respond to conditions that are distinct from the conditions sensed by AKT in the IIS pathway . In addition to mutations that reduce insulin signaling , various stress conditions , including thermal stress , oxidative stress , and starvation , can trigger translocation of DAF-16 to the nucleus ( Henderson and Johnson , 2001; Lin et al . , 2001 ) . Among the DAF-16 target genes are a wide range of stress response genes , including small heat shock proteins , a superoxide dismutase , catalases , and glutathione S-transferases ( Murphy et al . , 2003; Dong et al . , 2007 ) . Transcriptional activation of these target genes by nuclear DAF-16 helps the organism cope with stress , and daf-16 mutant worms survive stressful conditions less well than WT worms ( Wolff et al . , 2006 ) . To test whether UNC-43 is required for transmitting stress signals to DAF-16 , we crossed DAF-16::6xHis::GFP into unc-43 ( null ) and compared these animals to WT animals carrying the same transgene . At 20°C , DAF-16::6xHis::GFP displayed a cytoplasmic pattern in either WT or unc-43 ( null ) animals . When these worms were transferred to 28°C , DAF-16::6xHis::GFP accumulated in the nucleus in WT worms but remained cytoplasmic in unc-43 ( null ) animals ( Figure 6G ) . Similarly , starvation and paraquat , a chemical that generates reactive oxygen species ( ROS ) inside the cells , caused DAF-16 to translocate to the nucleus in WT but not unc-43 ( null ) worms ( Figure 6G ) . The results demonstrated that unc-43 is required for nuclear accumulation of DAF-16 in response to different types of stress . The site of DAF-16 that is phosphorylated by CAMKII is different from the site that is phosphorylated by AKT . Moreover , phosphorylation by AKT and CAMKII has opposite effects on DAF-16 activity ( Figures 5 and 6 and Lin et al . , 2001 ) . Thus , the IIS pathway ( DAF-2 ) and the CAMKII pathway might work in parallel . In agreement with this hypothesis , reduced IIS and increased phosphorylation of DAF-16 S286 , either by unc-43 ( gf ) or tax-6 ( lf ) , displayed an additive effect on lifespan ( Figure 7A–B , Figure 7—source data 1A ) . These two pathways , however , also seem to crosstalk . First , an unc-43 null allele shortened the daf-2 lifespan by 31% ( Figure 7C , Figure 7—source data 1B ) , indicating that unc-43 is required for full lifespan extension by daf-2 ( e1370 ) . This result suggests that part of the signaling from DAF-2 is mediated by UNC-43 . However , phosphorylation of DAF-16 S286 seems nonessential for the longevity of daf-2 animals ( Figure 7—figure supplement 1 , Figure 7—source data 1C ) , suggesting that an additional UNC-43 target ( s ) is involved in DAF-2 signaling . Second , in the constitutively active unc-43 ( gf ) mutant , which has increased phosphorylation on DAF-16 S286 ( Figure 5D ) , phosphorylation by AKT on T240 is greatly reduced ( Figure 5E ) . Therefore , in long-lived unc-43 ( gf ) animals , phosphorylation on these two residues appears to be coordinated , but it is unclear how this coordination occurs . The AKT kinase activity may be reduced in unc-43 ( gf ) , or phospho-S286 DAF-16 may be a poor substrate for AKT . In either case , dampened phosphorylation on the AKT site may be expected to prevent DAF-16 from forming a complex with the 14-3-3 protein , which has been shown to bind to and sequester AKT-phosphorylated FOXO ( DAF-16 or mammalian FOXO3 ) in the cytoplasm ( Brunet et al . , 1999; Berdichevsky et al . , 2006; Li et al . , 2007 ) . Consistently , we found that unc-43 ( gf ) reduced the amount of DAF-16-associated 14-3-3 without affecting the total amount of 14-3-3 ( Figure 7—figure supplement 2 ) . It remains to be seen whether S286 phosphorylation directly or indirectly promotes DAF-16 nuclear accumulation . 10 . 7554/eLife . 00518 . 017Figure 7 . 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 ( e1370 ) ;unc-43 ( n498n1186 ) double mutant lived a significantly shorter life than daf-2 ( e1370 ) , while the unc-43 ( n498n1186 ) mutant had a WT-like lifespan . In ( A ) and ( B ) , p<0 . 001 for WT vs tax-6 ( null ) or unc-43 ( gf ) , unc-43 ( gf ) ;daf-2 ( RNAi ) vs . daf-2 ( RNAi ) or unc-43 ( gf ) , and tax-6 ( null ) ;daf-2 ( RNAi ) vs . daf-2 ( RNAi ) or tax-6 ( null ) . In ( C ) , p<0 . 001 for daf-2 vs . daf-2;unc-43 ( null ) , p=0 . 61 for unc-43 ( null ) vs . WT in a lifespan assay at 25°C . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01710 . 7554/eLife . 00518 . 018Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01810 . 7554/eLife . 00518 . 019Figure 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 hqEx366 ) . n ≥ 61 . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 01910 . 7554/eLife . 00518 . 020Figure 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 by WB using an antibody that recognizes both 14-3-3 proteins ( FTT-2 and PAR-5 ) in C . elegans . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 020 While IIS and CAMKII/Calcineurin signaling act largely in parallel with each other , it is unclear what would happen if one pathway was in a state to inhibit DAF-16 while the other pathway was in a state that would activate it . To genetically mimic this scenario , we combined daf-2 RNAi with either unc-43 ( null ) or tax-6 ( jh107 ) , a constitutively active tax-6 ( gf ) allele in which the autoinhibitory domain was deleted ( Lee et al . , 2004 ) . DAF-16::GFP was cytoplasmic in unc-43 ( null ) or tax-6 ( gf ) , but nuclear DAF-16::GFP was clearly visible above the cytoplasmic background in daf-2 ( RNAi ) , daf-2 ( RNAi ) ;unc-43 ( null ) , or daf-2 ( RNAi ) ;tax-6 ( gf ) mutants ( Figure 8A ) . We also combined daf-18 RNAi , which enhances IIS to inhibit DAF-16 ( Ogg and Ruvkun , 1998 ) , with unc-43 ( gf ) or tax-6 ( lf ) , which activates DAF-16 . In both cases , the nuclear accumulation induced by unc-43 ( gf ) or tax-6 ( lf ) was greatly diminished by daf-18 RNAi ( Figure 8B ) . Therefore , under conditions in which there is a conflict between IIS and CAMKII/Calcineurin signaling toward DAF-16 , the insulin signaling pathway appears to dominate . 10 . 7554/eLife . 00518 . 021Figure 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 daf-18 ( RNAi ) . ( C ) A model showing the regulation of DAF-16 ( FOXO ) by insulin signaling , UNC-43 ( CAMKII ) , and TAX-6•CNB-1 ( Calcineurin ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 021 Based on the experimental evidence above , we propose a model ( Figure 8C ) in which DAF-16 integrates hormonal signals and stress signals transmitted by the IIS pathway ( from DAF-2 to AKT ) and CAMKII ( UNC-43 ) , respectively . CAMKII and the counterbalancing phosphatase Calcineurin ( TAX-6•CNB-1 ) act in parallel and coordinately with the IIS pathway to regulate DAF-16 activity . Sequence analysis of FOXO proteins revealed that DAF-16 S286 , the CAMKII phosphorylation site , is widely conserved in nematode species , zebra fish , frogs , mice , and humans ( Figure 9A ) . It is equivalent to S298 of mouse FOXO3 ( mFOXO3 ) and S299 of human FOXO3 ( hFOXO3 ) ( Figure 9A ) , and may be the equivalent of S303 of human FOXO1 or S300 of mouse FOXO1 ( not shown ) . Such conservation suggests that mammalian FOXO proteins are likely regulated by CAMKII and Calcineurin . 10 . 7554/eLife . 00518 . 022Figure 9 . 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 , but it is found in many nematode FOXOs and a number of vertebrate FOXOs . The DAF-16 or mouse FOXO3 residues phosphorylated in vitro by UNC-43 or CAMKIIA are boxed , with amino acid positions shown above or below . GFP-tagged mouse CAMKIIA or CAMKIIB , or Myc-tagged human Calcineurin A was transfected either alone or with FLAG-tagged mouse FOXO3 into HEK293T cells . Immunoprecipitation of FLAG-FOXO3 pulled down both CAMKII isoforms ( B ) and Calcineurin ( C ) . ( D ) Two FOXO3 fragments P2 and P3 , corresponding to amino acids 154–259 and 259–409 , were phosphorylated in vitro by CAMKIIA in the presence of [32P]-γ-ATP . The input protein substrates are shown at the bottom . ( E ) Mass spectrum showing a FOXO3 peptide phosphorylated by CAMKIIA at S298 . ( F ) CAMKIIA phosphorylates FOXO3 at S298 in vivo . FLAG-FOXO3 immunoprecipitated from HEK293T cells co-transfected or not with CAMKIIA was digested with trypsin and subjected to mass spectrometry analysis . Spectral counts ( i . e . , number of observations ) of the indicated phospho-S/T are normalized to the total spectral counts of FLAG-FOXO3 . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 02210 . 7554/eLife . 00518 . 023Figure 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 9E . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 02310 . 7554/eLife . 00518 . 024Figure 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 FOXO3 at S253 , which was recognized using an antibody specific for S253 phosphorylation . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 02410 . 7554/eLife . 00518 . 025Figure 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 . Calcineurin markedly reduced the CAMKIIA-induced phosphorylation on P3 . ( B ) The P2 fragment of mouse FOXO3 that was phosphorylated by CAMKIIA in vitro and then incubated with Calcineurin after inactivation of the kinase . Calcineurin had no effect on mFOXO3 S252 phosphorylation as visualized by immunoblotting with a phospho-S252 specific antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 025 To determine if this is true , we first expressed a FLAG-tagged mouse FOXO3 together with a GFP-tagged mouse CAMKII alpha ( CAMKIIA ) or beta ( CAMKIIB ) isoform , or a Myc-tagged human Calcineurin A ( CnA ) in HEK293T cells , and found that CAMKII and CnA both interact with FOXO3 ( Figure 9B–C ) . Next , we asked whether CAMKII phosphorylates FOXO3 in vitro . Purified GST fusion proteins containing five non-overlapping fragments of FOXO3 ( Lehtinen et al . , 2006 ) were incubated with an active recombinant human CAMKIIA . CAMKIIA robustly phosphorylated the P2 and P3 fragments but not the others ( Figure 9D ) , placing the phosphorylation sites within aa 154–409 . Mass spectrometry and mutagenesis studies of the full-length mFOXO3 or the P2 and P3 fragments from the in vitro kinase assay mapped the CAMKIIA phosphorylation sites to S252 , S279 , and S298 ( not shown ) . The same sites were also identified from FLAG-mFOXO3 co-expressed with CAMKIIA in HEK293T cells ( Figure 9E , Figure 9—figure supplement 1 ) . However , only mFOXO3 S298 , which corresponds to DAF-16 S286 , had a significant increase in phosphorylation when cells were co-transfected with CAMKIIA ( Figure 9F ) . This shows that S298 is the principle CAMKIIA phosphorylation site in vivo . S252 in mouse FOXO3 or S253 in human FOXO3 , the counterpart of DAF-16 T240 ( Figure 9A ) , is a highly conserved AKT phosphorylation site ( Brunet et al . , 1999 ) . Using a specific antibody , we found that neither CAMKIIA nor Calcineurin regulated phosphorylation of endogenous human FOXO3 at S253 in HEK293T cells ( Figure 9—figure supplement 2 ) . Moreover , Calcineurin can reduce the phosphorylation of the FOXO3-P3 fragment ( containing S298 ) by CAMKIIA , but not that of the P2 fragment ( containing S252 ) ( Figure 9—figure supplement 3 ) . Taken together , these results demonstrate that mammalian CAMKII and Calcineurin regulate phosphorylation of FOXO3 at the same conserved site as in C . elegans ( S298 in mouse FOXO3 and S286 in DAF-16 ) . A recent study by Ozcan et al . shows that four non-AKT phosphorylation sites are involved in the activation of murine FOXO1 by CAMKIIγ , likely through the p38 MAP kinase , in glucagon-stimulated hepatocytes ( Ozcan et al . , 2012 ) . Serine 300 of mouse FOXO1 , the equivalent of S298 of mouse FOXO3 , is not among them ( S246 , S295 , S467 , and S475 ) . To find out whether the CAMKIIA isoform phosphorylates FOXO1 in the same way as it does FOXO3 , we expressed a FLAG-tagged human FOXO1 in HEK293T cells co-transfected or not with CAMKIIA . Mass spectrometry analysis identified three CAMKIIA-regulated phosphorylation sites equivalent to mouse FOXO1 S295 , S467 , and S475 ( not shown ) . This agrees with the findings by Ozcan et al . and suggests that CAMKII directly phosphorylates FOXO3 but not FOXO1 in mammals . Lastly , we determined whether CAMKII and Calcineurin regulate the transcriptional activity of FOXO3 . Mammalian CAMKII can be activated by autophosphorylation at threonine 286 ( Mukherji et al . , 1994; Rich and Schulman , 1998 ) . Mutation of this residue to A or D results in a kinase dead or constitutively active form of CAMKII , respectively ( Fong et al . , 1989 ) . Using a luciferase reporter assay , we found that the transcriptional activity of FOXO3 was indeed stimulated by CAMKIIA in a way that required S298 of FOXO3 ( Figure 10A ) and T286 of CAMKIIA ( Figure 10B ) , and further enhanced by the constitutively active CAMKIIA mutant ( Figure 10C ) . Consistently , expression of Calcineurin significantly inhibited the transcriptional activity of FOXO3 ( Figure 10D ) . From these results , we conclude that CAMKIIA and Calcineurin oppose each other in regulating the transcriptional activity of mouse FOXO3 through phosphorylation or dephosphorylation at S298 . This mechanism is conserved from C . elegans to mammals . 10 . 7554/eLife . 00518 . 026Figure 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 activity relative to the empty vector transfection is plotted ( ***p<0 . 0001 , Student’s t-test , n = 3; N . S . for not significant ) . ( A ) Co-transfection of CAMKIIA stimulated the transcriptional activity of WT FOXO3 but not the FOXO3 ( S298A ) mutant . ( B ) WT but not an inactive T286A mutant CAMKIIA , transcriptionally activated FOXO3 . ( C ) A constitutively active T286D mutant CAMKIIA further enhanced FOXO3 activity compared to WT CAMKIIA . ( D ) FOXO3 was transcriptionally inhibited by Calcineurin A , and further inhibited if Calcineurin A and Calcineurin B were both expressed . DOI: http://dx . doi . org/10 . 7554/eLife . 00518 . 026
Our data suggest that CAMKII ( UNC-43 ) and Calcineurin ( TAX-6•CNB-1 ) , by phosphorylating or de-phosphorylating serine 286 of DAF-16 , extend or shorten the lifespan of C . elegans . The direct , antagonistic nature of these two enzymes also raised an interesting possibility that , with their opposing effects on DAF-16 and a common activating signal ( Ca2+/CAM ) , UNC-43 and TAX-6•CNB-1 would constitutively negate each other and have essentially no control of DAF-16 . However , this outcome most likely does not occur because of the difference in the spatial distribution and temporal activation profiles of the two enzymes . TAX-6 and CNB-1 , similar to DAF-16 , are expressed in most cells in C . elegans ( Ogg et al . , 1997; Bandyopadhyay et al . , 2002; Kuhara et al . , 2002; Dong et al . , 2007 ) . In contrast , unc-43 promoter-driven GFP was only detected in neurons and the intestine ( Hunt-Newbury et al . , 2007 ) . We also detected UNC-43 primarily in the nervous system ( not shown ) . At the subcellular level , the TAX-6::GFP fusion protein is distributed diffusely throughout the cytoplasm and inside the nucleus ( Kuhara et al . , 2002; Dong et al . , 2007 ) . In contrast , the CFP::UNC-43 fusion protein is concentrated on perinuclear structures and clusters in neurites ( Umemura et al . , 2005 ) . Temporally , Ca2+-activated CAMKII phosphorylates another molecule of CAMKII at T286 to convert it to a Ca2+/CAM-independent kinase , prolonging the kinase activity after the initial activation ( Erickson et al . , 2011 ) . CAMKII can also be activated by ROS at low Ca2+ concentrations ( Erickson et al . , 2011 ) . In mammalian cells , inhibition of Calcineurin by cytoplasmic CAMKII and inhibition of CAMKII by Calcineurin have both been reported ( MacDonnell et al . , 2009; Kubokawa et al . , 2011 ) , suggesting that the balance is intricately maintained . Therefore , in the case of the simultaneous activation of CAMKII and Calcineurin by Ca2+/CAM , either the kinase or the phosphatase could dominate the regulation of DAF-16 either locally ( e . g . , cytoplasm vs nucleus ) or for certain time windows . This balance likely fine-tunes the DAF-16 activity in response to a changing environment . It is likely that UNC-43/Calcineurin is responsible for activating DAF-16 when worms experience a variety of stress signals , including heat stress , oxidative stress , or prolonged starvation ( Figure 6G ) . Food deprivation acts through UNC-43 to suppress spontaneous sex-muscle contraction in C . elegans males ( LeBoeuf et al . , 2007 ) . Reported in HEK293T cells , human FOXO3 is phosphorylated at non-AKT sites under stress conditions such as heat shock or H2O2 treatment ( Brunet et al . , 2004 ) . ROS-induced oxidation of two methionine residues activates CAMKII , and this activation can occur under conditions of low Ca2+ ( Erickson et al . , 2011 ) . Thus , CAMKII is perfectly equipped to transmit ROS signaling and calcium signaling , and phosphorylation of FOXO3 by CAMKII may play a highly conserved role in the stress response , from worms to humans . To date , DAF-16 is the only biochemically and genetically proven substrate of TAX-6•CNB-1 , but it is not the only substrate involved in lifespan regulation by Calcineurin . Inactivation of CRTC-1 , a coactivator of CREB , contributes to the longevity of tax-6 ( lf ) animals ( Mair et al . , 2011 ) . Hence , CRTC-1 is an excellent candidate , although it has not been shown that TAX-6•CNB-1 directly dephosphorylates CRTC-1 . There may be additional substrates . Regardless , the discoveries made thus far jointly provide a good explanation for the large but incomplete suppression of tax-6 ( lf ) longevity by daf-16 ( null ) ( Dong et al . , 2007 ) . Post-transplant new-onset diabetes mellitus ( NODM ) is a frequent and serious complication for organ transplant patients . One of the NODM risk factors is the use of Calcineurin inhibitors , such as cyclosporin A and FK506 , which suppress immune-rejection of donor organs ( Heisel et al . , 2004 ) . In light of this work , we propose that sustained activation of FOXO3 resulting from chronic inhibition of Calcineurin may cause NODM in transplant patients . Given the conservation and broad tissue distribution of CAMKII , Calcineurin , and FOXO3 , the regulatory mechanism uncovered in this study likely plays a role in many physiological functions .
All strains were cultured at 20°C following standard protocols unless otherwise indicated ( Brenner , 1974 ) . Double mutants were made using standard genetic methods , and the genotypes were confirmed using PCR or PCR followed by sequencing . The strains and oligos used in the study are listed in Supplementary file 1A , B , respectively . The following antibodies were purchased: Mouse anti-Actin ( Sigma-Aldrich , St Louis , MO ) , Mouse anti-tubulin ( Sigma-Aldrich ) , mouse anti-CAM ( Millipore , Billerica , MA ) , mouse anti-FLAG ( Sigma-Aldrich ) , anti-FLAG M2 Affinity Gel ( Sigma-Aldrich ) , rabbit anti-GFP ( Abmart , Shanghai , China ) , mouse anti-GFP ( Roche , Basel , Switzerland ) , rabbit anti-GFP ( Invitrogen , Carlsbad , CA ) , rabbit anti-GST ( GeneSci , Beijing , China ) , monoclonal anti-HA agarose ( Sigma-Aldrich ) , rabbit anti-mFOXO3 ( phospho-S252 ) /hFOXO3 ( phospho-S253 ) ( Cell Signaling , Danvers , MA ) , Mouse anti-Myc ( Santa Cruz Biotechnology , Dallas , TX ) , goat anti-mouse IgG HRP ( Jackson Immuno Research , West Grove , PA ) , and goat anti-rabbit IgG HRP ( BaiHuiZhongYuan , Beijing , China ) . Rabbit α-HA and monoclonal mouse α-DAF-16 antibodies were made at the antibody center of the NIBS , Beijing . A single-chain anti-GFP antibody was produced as described before ( Kubala et al . , 2010 ) and cross-linked to NHS-activated Sepharose 4 Fast Flow beads ( GE Healthcare , Piscataway Township , NJ ) . We called this GBP beads . To produce the DAF-16 antibody , full-length DAF-16b was cloned into the His-tag vector pET-28a , expressed in Escherichia coli BL21 , and purified by Ni-NTA resin ( Qiagen ) . Rabbit α-DAF-16b ( pT240 ) and α-DAF-16b ( pS286 ) antibodies were custom made and affinity purified at Abmart ( Shanghai , China ) using the synthetic DAF-16 phospho-peptides ‘RERSN ( pT ) IETTT-C’ and ‘SIQTI ( pS ) HDLYD-C , ’ respectively . A Rabbit polyclonal anti-human 14-3-3 antibody that recognizes both C . elegans 14-3-3 proteins was a gift from Dr Yamei Wang ( Xiamen University , China ) . The daf-16 constructs were subcloned and further modified from a Pdaf-16::daf-16b::gfp construct described previously ( the daf-16b isoform used to be called daf-16a2 ) ( Henderson and Johnson , 2001 ) . The GST-CNB-1 and TAX-6 bacterial expression constructs were modified from the GST-CNB-1 and GST-TAX-6 plasmids reported before ( Bandyopadhyay et al . , 2002 ) . The unc-43 expression constructs were made from unc-43 cDNAs that were amplified from a C . elegans cDNA library . The constructs expressing full-length or truncated mouse FOXO3 have been described ( Lehtinen et al . , 2006 ) , and the FOXO3-P2-S252A mutant was made by site-directed mutagenesis . The CAMKIIA and CAMKIIB expression constructs have described ( Gaudillière et al . , 2004 ) . The Myc-CnA and Myc-CnB expression constructs under the CMV promoter were subcloned from ‘pET15b CnA CnB’ ( Addgene ) . The FKRE-luciferase expression vector was kindly provided by Dr Azad Bonni ( Harvard Medical School ) . GST-CNB-1 and TAX-6 were co-expressed in E . coli BL21 and affinity purified using glutathione Sepharose ( GE Healthcare ) in PBS containing 0 . 5% NP-40 following standard protocols . GST-DAF-16 ( N ) and GST-DAF-16 ( N-F ) were purified in the same manner . Recombinant GST-UNC-43 was purified as described above but using a buffer containing 50 mM Tris-HCl , pH 7 . 6 , 150 mM NaCl , 0 . 5% NP-40 . Recombinant 6xHis-DAF-16 and 6xHis-DAF-16-6AM were expressed in BL21 and affinity purified using Ni-NTA agarose resin ( Qiagen ) in buffer containing 20 mM HEPES , pH 7 . 5 , 150 mM NaCl , and 0 . 5% NP-40 . His-tagged proteins were eluted off beads by adding 250 or 500 mM imidazole to the buffer after a 30 mM imidazole wash . Eluted proteins underwent buffer exchange and were concentrated using Centricon purifying units ( Millipore ) with 10-kDa ( for TAX•GST-CNB-1 ) or 50-kDa ( for other proteins ) cutoffs to 0 . 5–1 . 0 mg/ml protein in storage buffer ( 20 mM HEPES , pH 7 . 5 , 150 mM NaCl , 0 . 5% NP-40 , 10% glycerol ) ; the resulting solutions contained >1 mM glutathione or >5 mM imidazole . Bovine Calmodulin and human Calcineurin were purchased from Merck . Active human GST-CAMKIIA was ordered from Sigma . Recombinant mFOXO3 P1-P5 fragments were purified as described ( Lehtinen et al . , 2006 ) . Lifespan assays were carried out at 20°C unless otherwise indicated . To synchronize worms , twenty adult worms were allowed to lay eggs on NGM plates for 4 hr before being removed . After the progeny grew to the L4 stage , they were transferred to new plates ( 10 worms/plate ) . At least ten plates ( 100 worms ) were used for the lifespan assay of each strain or RNAi treatment . Worms were transferred to fresh plates every 2 days until they ceased laying egg , after which worms were transferred to fresh plates every week . For lifespan assays that involved or would be compared with the unc-43 ( gf ) mutant , 50 μg/ml FUDR was added to the plates to prevent its Egl ( egg-laying defective ) phenotype from interfering with lifespan measurement . In these experiments ( Figure 2—source data 1B; Figure 2—source data 1C; Figure 3—source data 1; Figure 6—source data 1A; Figure 6—source data 1C; Figure 7—source data 1A ) , worms were transferred to fresh plates every 4 days until death . Live worms were scored every 2 days . Worms were considered dead if they failed to respond to gentle touches with a worm pick on the head and tail . Worms that had internally hatched larvae ( ‘bagged’ ) or ruptured vulvae ( ‘exploded’ ) or crawled off the agar surface were censored . SPSS ( Statistical Package for the Social Sciences ) software was used for the statistical analysis of the lifespan data , and the log-rank method was used to calculate p values . RNAi assays were performed at 20°C using the feeding method as previously described ( Kamath et al . , 2003 ) . Worms were fed RNAi bacteria from the time of hatching unless otherwise indicated . E . coli HT115 ( DE3 ) transformed with pAD12 , an empty RNAi vector ( Arantes-Oliveira et al . , 2002 ) , was used as a control . pTL13 is an RNAi construct targeting unc-43 , which we generated by inserting a 332-bp cDNA fragment of unc-43 into the multiple cloning sites of pAD12 . The other RNAi bacterial strains were obtained from the Ahringer RNAi library . To examine the interaction between DAF-16 and TAX-6 , 6xHis-DAF-16 was incubated with purified TAX-6•GST-CNB-1 complex in the presence of 2 μg CAM and 30 μl pre-washed Ni-NTA agarose resin ( Qiagen ) in binding buffer A ( 50 mM Tris , pH 8 . 0 , 100 mM NaCl , 4 mM CaCl2 , 2 mM MgCl2 , 0 . 5% NP-40 ) at 4°C for 2 hr . The beads were then washed with washing buffer A ( 50 mM Tris , pH 8 . 0 , 100 mM NaCl , 4 mM CaCl2 , 2 mM MgCl2 , 0 . 5% NP-40 , 30 mM imidazole ) and eluted with SDS loading buffer . Samples were resolved by SDS-PAGE and stained with Coomassie blue . For DAF-16 and UNC-43 binding , 6xHis-DAF-16 was incubated with purified GST-UNC-43 and CAM in the presence of 30 μl pre-washed Ni-NTA agarose in binding buffer A without MgCl2 . After binding , beads were washed with washing buffer A without MgCl2 and eluted with SDS loading buffer . The samples were resolved by replicate SDS-PAGE gels , one for Coomassie blue or silver staining , and the other for western blots with anti-GST , anti-DAF-16 or anti-CAM antibodies . To map the TAX-6 binding site on DAF-16 , 5 µg of GST or GST-tagged DAF-16 fragments were incubated with TAX-6::GFP-containing worm lysate ( ∼15 µg/µl proteins in 300 μl of 20 mM Tris , pH 8 . 0 , 1% NP-40 , 10% glycerol , 1x Protease Inhibitor Cocktail , EDTA free , Roche ) and pre-washed glutathione beads at 4°C for 1 hr . The beads were washed three times and eluted with 1x SDS loading buffer . Mixed-stage worms were cultured at 20°C on five 100-mm HG plates seeded with OP50 , harvested , and washed with M9 buffer to yield 200–500 μl of packed worms . Then , 200 μl of packed worms was mixed with 200 μl of 2x lysis buffer A , B , or C ( see below ) and 800 μl of 0 . 5-mm diameter glass beads . The mixture was lysed using FastPrep-24 ( MP Biomedicals ) at 6 . 5 m/s , 20 s/pulse × 3 pulses with 5-min intervals on ice . Worm lysates were cleared by centrifugation at 13 , 000 rpm for 30 min . For anti-GFP and anti-FLAG IPs , the supernatant was incubated with GBP beads or anti-FLAG M2 beads for 1–2 hr . For the phospho-DAF-16 IP , the supernatant was incubated with 10 μl of the antibody specific for DAF-16 ( pT240 ) or DAF-16 ( pS298 ) and 30 μl of pre-washed protein A and protein G beads ( mixed at 1:1 ) for 2 hr . After incubation with the lysates , the beads were washed 2–3 times with 1x lysis buffer , 5 min each time , and boiled in 2x SDS loading buffer for western blot analysis . All steps , from making the lysates to eluting the beads , were performed at 4°C . 1x lysis buffer A ( for IP of DAF-16 and TAX-6 ) : 20 mM Tris pH 8 . 0 , 1% NP-40 , 10% glycerol , 2 mM EDTA , 1x PIC ( Protease Inhibitor Cocktail , EDTA free; Roche ) . 1x lysis buffer B ( for IP of DAF-16 and UNC-43 ) : 50 mM Tris , pH 8 . 0 , 150 mM NaCl , 0 . 1% NP-40 , 10% glycerol , 1x PIC , 1x PhosSTOP ( Roche ) . 1x lysis buffer C ( for IP of phospho-DAF-16 ) : 50 mM Tris , pH 7 . 6 , 150 mM NaCl , 0 . 1% SDS , 0 . 25% sodium deoxycholate , 1% NP-40 , 2 mM EDTA , 1x PIC , 1x PhosSTOP . Immunoprecipitation of FLAG-FOXO3 from HEK293T cells was performed as described ( Xie et al . , 2012 ) . 2 μg of purified recombinant GST-UNC-43 ( or 5 μg of purified active human GST-CAMKIIA ) was incubated with 2 μg of CAM and 2–5 μg of purified 6xHis- or GST-tagged DAF-16 ( or GST-FOXO3 ) in the kinase reaction buffer ( 50 mM PIPES , pH 7 . 0 , 10 mM MgCl2 , 4 mM CaCl2 , 100 μM ATP , 1x PIC ) in the presence or absence of [32P]-γ-ATP at 30°C for 15 min . Proteins were resolved by SDS PAGE and analyzed by autoradiography or WB with anti-DAF-16 ( pT240 ) or anti-DAF-16 ( pS286 ) antibodies . The kinase reactions , performed as described above , were shifted to 60°C for 10 min to inactivate UNC-43 or CAMKIIA . Then the samples were incubated or not with 5 μg of TAX-6•GST-CNB-1 or Calcineurin in phosphatase buffer ( 50 mM Tris , pH 7 . 5 , 100 mM NaCl , 6 mM MgCl2 , 1 mM CaCl2 , 1x PIC ) in the presence of 2 μg CAM at 30°C for 30 min . The sample were resolved by SDS-PAGE and analyzed by autoradiography or by WB with antibodies specific for DAF-16 ( pT240 ) , DAF-16 ( pS286 ) , or mFOXO3 ( S252 ) /hFOXO3 ( S253 ) . Worms were cultured at 20°C on NGM plates from eggs laid within a 4-hr period and imaged at the L4 stage unless otherwise indicated . As soon as worms were removed from incubation , they were mounted on slides and imaged immediately . For the starvation challenge , approximately 100 L4 worms were placed on an empty plate and kept at 20°C for 20 hr before imaging . For the heat stress challenge , well-fed L4 worms on bacterial food were shifted to 28°C for 2 hr . Parallel samples were maintained at 20°C on food as controls . All GFP and DIC images were taken using a Zeiss Axio Imager M1 microscope at 400-fold magnification . Coomassie blue-stained bands corresponding to in vitro phosphorylated DAF-16 or FOXO3 fragments , or FLAG-FOXO3 immunoprecipetated from 293T cells were destained and in-gel digested with trypsin . LC-MS/MS analyses of the resulting peptides were carried out on an LTQ-Orbitrap ( ThermoFisher Scientific ) or LTQ-Orbitrap Velos ( ThermoFisher Scientific ) as described before with slight modifications ( Li et al . , 2011 ) . CID MS2 with neutral-loss triggered MS3 spectra were collected on LTQ-Orbitrap and HCD MS2 spectra were collected on LTQ-Orbitrap Velos . The MS2 or MS3 data were searched against the C . elegans protein database ( for DAF-16 ) or the NCBI mouse protein database ( for FOXO3 ) . The pLabel software was used for spectral labeling ( Yang et al . , 2012 ) . HEK293T cells were maintained in DMEM medium supplemented with 10% fetal bovine serum ( FBS ) at 5% CO2 . Transfection was carried out using Lipofectamine 2000 ( Invitrogen ) , and the luciferase assays were carried out using the Dual-Luciferase assay kit ( Promega ) .
The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16 , the ortholog of the FOXO transcription factors of mammalian cells . DAF-16 is phosphorylated by the AKT kinases , preventing its nuclear translocation . Calcineurin ( PP2B phosphatase ) also limits the lifespan of C . elegans , but the mechanism through which it does so is unknown . Herein , we show that TAX-6•CNB-1 and UNC-43 , the C . elegans Calcineurin and Ca2+/calmodulin-dependent kinase type II ( CAMKII ) orthologs , respectively , also regulate lifespan through DAF-16 . Moreover , UNC-43 regulates DAF-16 in response to various stress conditions , including starvation , heat or oxidative stress , and cooperatively contributes to lifespan regulation by insulin signaling . However , unlike insulin signaling , UNC-43 phosphorylates and activates DAF-16 , thus promoting its nuclear localization . The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1 , leading to DAF-16 inactivation . Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin .
Although aging might seem to be a passive process—resulting simply from wear and tear over a lifetime—it can actually be accelerated or slowed down by genetic mutations . This phenomenon has been most thoroughly studied in the nematode worm , Caenorhabditis elegans . Normally , this worm lives for just two or three weeks , but genetic mutations that reduce the activity of certain enzymes in a series of biochemical reactions known as the insulin/IGF-1 signalling pathway can extend its lifespan by up to a factor of ten , and similar effects have been seen in flies and mice . Lifespans can also be increased by blocking other signalling pathways or restricting the intake of calories . This increase in lifespan associated with the insulin/IGF-1 signalling pathway is known to involve a protein called DAF-16 and two kinases called AKT-1 and AKT-2 . Under normal conditions the AKT kinases add several phosphate groups to the DAF-16 , which prevents it from travelling to the nucleus of the cell . However , when genetic techniques are used to block the insulin/IGF-1 signalling pathway , the AKT kinases are unable to add the phosphate groups; this leaves the DAF-16 free to enter the nucleus , where it activates a network of genes that promotes longevity . In addition to kinases , the insulin/IGF-1 signalling pathway also involves enzymes called phosphatases that remove the phosphate groups from other proteins . In particular , a phosphatase called calcineurin is known to be involved in the regulation of lifespan , but the details of this process are not fully understood . Now , Tao et al . have carried out a series of genetic and biochemical experiments to determine how phosphatases exert their influence on aging . The results show that calcineurin targets DAF-16 , the same protein that is targeted by the AKT kinases . Moreover , another kinase also targets DAF-16 when the worm is exposed to heat , starvation or some other form of stress: this kinase , which is not involved in the insulin/IGF-1 signalling pathway , is called CAMKII . Tao et al . show that these kinases act on DAF-16 in different ways: CAMKII activates it by adding the phosphate group at a specific site known as S286 , whereas the AKT kinases deactivate DAF-16 because they add phosphate groups at different sites , thereby preventing it from entering the nucleus . Calcineurin neutralizes the effect of CAMKII by removing the phosphate group at S286 to deactivate the DAF-16 . In addition to shedding new light on the regulation of lifespan in C . elegans , the new results could improve our understanding of aging in humans , and also the development of diabetes and other age-related diseases , because the equivalent molecules in mammalian cells are regulated in similar ways .