Fig 1.
The effects of secreted Klotho on rotenone (ROT)-induced activation of p38 MAPK and Trx/ASK1 complex levels in HEK 293 cells.
(A) Plots of time course of ROT-mediated activation of p38-MAPK. Experiments were done at least twice and deviations are shown ± SEM. A representative Western blot is shown beneath plot. (B) Depiction of Trx/ASK1 complex levels in HEK 293 cells after pre-incubation with either control buffer alone or 200 pM Klotho/20 mM NAC for 40 min before adding the 5 μM ROT to induce oxidative stress. ASK1 was immunoprecipitated (IP) with rabbit polyclonal ASK1 antibody and the co-precipitated Trx was revealed by Western blot. A representative Western blot of the samples is shown beneath. *p< 0.05 between ROT and Klotho + ROT. (C) Plots of p38 MAPK activation levels in HEK 293 cells treated with either Klotho or NAC as described in section B, with a representative Western blot. *p< 0.05 between ROT and Klotho + ROT. (D) Total lysate levels of Trx prior to IP. Where phosphorylation was studied, the same blot was stripped with RestoreTM stripping buffer (Thermo Scientific) and re-probed with total p38 MAPK antibody for protein normalization. Digitized values of the WB of are shown in S1 and S2 Tables.
Fig 2.
Time course of Klotho-mediated phosphorylation of endogenous 14-3-3ζ (Ser-58) effect on 14-3-3ζ monomer levels in HEK 293 cells.
(A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demonstrated by adding 200 pM of the recombinant protein to the cultured cells at the indicated times. Peak phosphorylation times (i.e. 30–45 min) are within the range where the Trx/ASK1 complex protection against oxidative stress occurred (Fig 1). Data are reported as ± SEM. (B) Plot of 14-3-3ζ monomer level in HEK 293 cells treated with either secreted Klotho or buffer control for 40 min. Shown beside plot is a native Western blot of the samples as described in Materials and Methods. The 14-3-3ζ antibody recognizes a predominant ~30 kDa protein band representing the expected size of the monomer. Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB of monomer levels are shown in S3 Table.
Fig 3.
Mitigating effect of soluble Klotho on endogenous 14-3-3ζ and Trx complex formation.
(A) Levels of Trx bound to endogenous 14-3-3ζ in HEK 293 cells treated with either secreted Klotho, or NAC, or buffer control for 40 min as described in text. *p< 0.05 between control buffer and Klotho treated samples. The Western blot profile is shown beneath. (B) Levels of Trx bound to GST-14-3-3ζ wild type (Wt) and dimerization deficient mutant (Dm). Samples were treated with either buffer only or secreted Klotho for 40 min. *p< 0.05 between Wt and Dm. Shown beneath is a corresponding representative Western blot. Digitized values of the WB are shown in S4 Table.
Fig 4.
siRNA knockdown of 14-3-3ζ expression in HEK 293 cells.
A time dependent plot of levels of 14-3-3ζ expression following siRNA knockdown of the 14-3-3ζ in HEK 293 cells. Cells were seeded at either 1.0 or 2.0 x106cells/well in 6-well culture dishes. For 14-3-3ζ targeting, three independent Stealth RNA duplex primers (Life Technologies) were pooled and used at a final concentration of 100 nM. Residual 14-3-3ζ expression levels were determined at 48, 72 and 96 h post transfection. Beneath the plots are Western blots showing the extent of 14-3-3ζ depletion (optimal at 72 h) at 1.0 x 106 cells/well.
Fig 5.
Effect of 14-3-3ζ knockdown on Klotho signaling via ASK1 and p38 MAPK pathway.
(A) Plots of phosphorylation levels of p38 MAPK in 14-3-3ζ-depleted HEK 293 cells. Cells were pre-treated with either 200 pM secreted Klotho or 20 mM NAC for 40 min before the addition of 5 μM ROT. Level of the phosphorylated protein was measured by Western blot using a phospho-14-3-3ζ (Ser-58) antibody. The same membrane was stripped and re-probed with the total 14-3-3ζ antibody to normalize for protein load. (B) Levels of Trx bound to ASK1 in 14-3-3ζ-depleted HEK 293 cells treated with secreted Klotho, or NAC, or buffer control for 40 min as described in text. The Western blot profile is shown beneath.
Fig 6.
Non-reducing 2D-GE Western blot analyses of ASK1 signaling complex in HEK 293 cells.
A non-reducing 2D-GE that preserves endogenous disulfide linkage was performed on Klotho-treated and buffer-control cells as described in the text. Images in A (buffer treated) or B (Klotho treated) are representative Western blots probed with either Trx antibody or stripped and re-probed with (i) ASK1 and/or (ii) 14-3-3ζ. Closed circled spots indicate free Trx and/or 14-3-3ζ proteins based on pI and/or molecular size. The dotted rectangular spots represent ASK1-bound complexes with Trx and/or 14-3-3ζ and other proteins cross-reactive with the Trx antibody that were more abundant in Klotho treated cells than in the buffer control.
Fig 7.
Effect of disulfide reducing agents on the dissociation of Trx from Trx bound ASK1 and/or 14-3-3ζ complex.
The ASK1 signaling complex was co-IP using ASK1 or 14-3-3ζ antibody. Chemical dissociation of co-precipitated Trx from the complex was monitored in the presence or absence of various disulfide reducing agents in SDS- conditioned buffer as described in the text. DTT, dithiothreitol; 2-ME, 2-mercaptoethanol; THP, tris(hydroxypropyl)phosphine. The experiment was repeated at least twice and a representative Western blot is shown. A representative full blot image showing higher molecular weight protein complexes is shown in S1 Fig
Fig 8.
iCE analyses of ASK1 signaling complex interactions.
Cells were either pretreated with buffer only or secreted Klotho for 40 min before adding 5 μM ROT to induce oxidative stress. Clear lysates were co-IP with antibody against ASK1 and the eluted protein complex was subjected to immuno-CE fractionation as described in the text. (A) Immuno-CE profile of eluted complex of buffer-only-treated control cells originally separated within pH 4–8 (pI range 5.5–6.6 is shown here for clarity) showing overlapping protein signals (arrow) within the pI range 6.25–6.30 comprising the ASK1/14-3-3ζ/Trx complex assembly. Signals belonging to individual proteins dissociated from the triad were also found within the pH 4–8. Embedded is a software generated gel view of the complex within the specified pI range. Individual pIs of the proteins are indicated in parenthesis. (B) Profile of eluted complex from cells treated with ROT. Overlapping signals within pIs 6.29–6.30 (arrowed) are those of the 14-3-3ζ/Trx complex. ASK1 signal was undetectable in the presence of ROT. Also shown is the gel view within the pI 5.5–6.6 range. (C) Profile of eluted complex from cells pretreated with secreted Klotho prior to ROT treatment. Overlapping signals within the pI 6.12–6.15 range (arrow) are the protected triad ASK1/14-3-3ζ/Trx complex. Shifts in pI of the complex to acidic end are noticeable.
Fig 9.
Model describing oxidative stress response in Klotho responsive cells and tissues.
(1–2) In normal unstressed cells and tissues, 14-3-3ζ and thioredoxin cooperate with other cytosolic proteins to inhibit the ASK1 signaling complex with concomitant downstream reduction in MAPKs phosphorylation level. (3–6) In an oxidizing environment generated by reactive oxygen species (ROS) via mitochondrial electron transport chain dysfunction induced by rotenone, ASK1 dissociates from thioredoxin/14-3-3ζ, becomes phosphorylated (activating ASK1 signaling compex) and, in turn, phosphorylates downstream MAPKs, including p38. These cascades of events inhibit nuclear translocation of transcription factors such as Nrf2, leading to suppression of antioxidant and longevity-promoting genes. Puzzling is our observation that 14-3-3ζ and thioredoxin remained partially bound. (7–10) By contrast, 14-3-3ζ interaction with the ASK1 signaling complex is consolidated by Klotho-dependent phosphorylation of the 14-3-3ζ at Ser-58, promoting monomer formation; 14-3-3ζ monomerization may increase its interaction with the inhibitory ASK1 by forming a stable complex. This activity is expected to facilitate Nrf2 nuclear localization promoting expression of antioxidant and longevity-promoting genes.