A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation

Abstract

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research.

Figure 1

pptr-1, a regulatory subunit of the PP2A holoenzyme, was identified as a top candidate in a directed RNAi screen to identify serine/threonine phosphatases that regulate the IIS pathway. A) The different families and classes of the phosphatases included in the RNAi screen. B) A schematic representation of the RNAi screen. All the assays were performed in triplicate. C) The top two candidates that dramatically suppressed daf-2(e1370) dauer formation at 20°C (fem-2, and pptr-1). Both fem-2 and pptr-1 RNAi were able to suppress daf-2 dauer formation to a similar level as daf-18 RNAi. Error bars indicate the standard deviations among the different RNAi plates within one experiment. Data shown [% Dauers ± Std. Dev. (n)] are from one representative experiment. D) pptr-1 is the only PP2A regulatory subunit family member that dramatically suppresses daf-2(1370) dauer formation. Error bars indicate the standard deviations among the different RNAi plates within one experiment. Data shown are from one representative experiment.

Figure 2

pptr-1 regulates lifespan, thermotolerance and fat-storage through the IIS pathway. Data shown are from one representative experiment. A) pptr-1 RNAi significantly reduces the lifespan of daf-2(e1370) mutants similar to daf-18 RNAi (mean life on vector RNAi is 33.9 ± 0.7 days (n=77), pptr-1 RNAi is 27.7 ± 0.9 days (n=63) p <0.0001and on daf-18 RNAi is 20.4 ± 0.6 days (n=40), p <0.0001) B) pptr-1 RNAi does not affect the lifespan of wild-type worms (mean lifespan on vector RNAi is 22.8 ± 0.4 days (n=61), pptr-1 RNAi is 21.9 ± 0.5 days (n=49). daf-18 RNAi reduces mean lifespan of wild type worms to 18.6 ± 0.3 days (n=48) p <0.0001). C) The thermotolerance of daf-2(e1370) worms is reduced by pptr-1 as well as daf-18 RNAi (mean survival of daf-2(e1370) worms at 37 °C on vector RNAi was 15.2 ± 0.7 hrs (n=34), whereas on pptr-1 RNAi the survival was 13.8 ± 0.5 hrs (p value< 0.006) (n=36) and 10.3 ± 0.7 hrs (p value< 0.0001) (n=29) on daf-18 RNAi. pptr-1 RNAi did not affect the thermotolerance of wild type worms; (mean survival was 9.8 ± 0.4 hrs on vector RNAi (n=32), 9.3 ± 0.3 hrs on pptr-1 RNAi (n=35) and 9.7 ± 0.4 hrs on daf-18 RNAi (n=32). D) Sudan black staining showing that pptr-1 RNAi reduces the increased fat storage of daf-2(e1370) worms, similar to daf-18 RNAi but has no effect on wild type fat-storage. Arrows indicate the pharynx. A representative picture from one of three independent experiments (n=30) is shown.

Figure 3

PPTR-1 co-localizes with AKT-1. akt-1∷gfp;pptr-1∷mC-flag, akt-2∷gfp;pptr-1∷mC-flag and sgk-1∷gfp;pptr-1∷mC-flag transgenic worms were mounted and visualized by fluorescence microscopy using Rhodamine (mCherry) and FITC (GFP) filters. PPTR-1 expression is observed mainly in the pharynx, vulva and spermatheca (A-C, mCherry). A) Expression of PPTR-1∷mC-FLAG (mCherry) and AKT-1∷GFP (GFP) in a akt-1∷gfp; pptr-1∷mC-flag strain. Expression of PPTR-1∷mC-FLAG overlaps with AKT-1∷GFP (Merge). B) PPTR-1∷mC-FLAG and AKT-2∷GFP colocalize in some tissues in a akt-2∷gfp; pptr-1∷mC-flag strain (Merge). C) SGK-1∷GFP and PPTR-1∷mC-FLAG do not colocalize in sgk-1∷gfp;pptr-1∷mC-flag transgenic worms (Merge). Arrows indicate the following tissues: p-pharynx, v-vulva, s-spermatheca, i-intestine

Figure 4

PPTR-1 interacts with and modulates AKT-1 phosphorylation. Data shown are from one representative experiment A) PPTR-1 directly interacts with AKT-1 in C. elegans. AKT-1∷GFP and MYO-3∷GFP were immunoprecipated (IP) using anti-GFP antibody and were analyzed by western blotting (WB) using anti-Ds-Red or anti-GFP antibodies. In addition, PPTR-1∷mC-FLAG was immunoprecipitated with anti-FLAG antibody and analysed by WB using using anti-Ds-Red or anti-GFP antibodies. Lysates were used for WB analysis. B) PPTR-1 overexpression reduces AKT-1 phosphorylation in C. elegans. AKT-1∷GFP and MYO-3∷GFP were immunoprecipitated from akt-1∷gfp, akt-1∷gfp;pptr-1∷mC-flag and myo-3∷gfp;pptr-1∷mC-flag followed by western blotting using pThr 350 or pSer 517 antibodies (upper panels). Total lysates were analyzed by western blotting (lower panels). Quantification of changes in AKT-1∷GFP phosphorylation upon PPTR-1 overexpression is shown below each lane. C) Knockdown of the mammalian B56β regulatory subunit by siRNA in 3T3-L1 adipocytes increases insulin-stimulated AKT phosphorylation at Thr 308. The 3T3-L1 adipocytes were transfected with scrambled (Scr), PP2Acα/β, B56α, B56 β or B56α/β siRNA. These cells were then treated with increasing concentrations of insulin and phosphorylation status of Akt was analyzed by western blotting using pThr 308 (left) and pSer 473 antibodies (middle). Total Akt antibody was used as a loading control (right). Quantification of fold changes in Akt phosphorylation is shown below each lane.

Figure 5

PPTR-1 regulates DAF-16 localization and activity. Data shown are from one representative experiment. A) Over-expression of PPTR-1 promotes DAF-16 nuclear translocation. On vector RNAi, DAF-16 is more enriched in the nucleus in a pptr-1∷mC-flag;daf-16∷gfp strain, compared to a daf-16∷gfp strain. This effect is specific to the functional transgene, as knocking down pptr-1∷mC-flag with mCherry RNAi decreases the extent of nuclear DAF-16. B) Overexpression of PPTR-1 significantly increases the lifespan of wild type worms. Mean lifespan of wild type worms is 23.9 ± 0.3 days (n=154), pptr-1∷mC-flag is 30.1 ± 0.5 days (n=202), p<.0001, and the unc-119(+); unc-119(ed3) control strain is 22.6 ± 0.3 days (n=145). C) In a daf-2(e1370);daf-16∷gfp strain, DAF-16 is enriched in the nucleus on vector RNAi, whereas on pptr-1 RNAi as well as daf-18 RNAi, DAf-16 is mostly cytosolic. D) pptr-1 RNAi affects DAF-16 transcriptional activity. sod-3 is one of the direct targets of DAF-16. pptr-1 RNAi reduces Psod-3∷GFP expression in a daf-2(e1370);Psod-3∷gfp(muIs84) strain, similar to daf-18 RNAi. E) Transcript abundance of known DAF-16 target genes decrease when daf-2(e1370) worms are grown on pptr-1 RNAi, similar to daf-18 RNAi, as detected by real-time PCR. F) Proposed model illustrating the role of PPTR-1 in the insulin/IGF-1 signaling pathway. Signals from DAF-2 are processed by a PI3-kinase pathway that leads to the phosphorylation and activation of downstream serine/threonine kinases such as PDK-1, AKT-1, AKT-2 and SGK-1. PPTR-1, the PP2A holoenzyme regulatory subunit, regulates the dephosphorylation and activation status of AKT-1 at T350. This in turn affects the nuclear translocation of DAF-16 and the expression of genes involved in lifespan, dauer formation, stress resistance and fat storage.