Figure 2. DAF-2 Signaling Inhibits Nuclear Accumulation of SKN-1 Directly, and Independently of DAF-16(A)
akt-1;
akt-2 or
sgk-1 RNAi allows
daf-16-independent accumulation of SKN-1 in intestinal nuclei. Expression of SKN-1op::GFP was monitored.
akt-1 or
akt-2 RNAi had a similar effect to combined
akt-1;
akt-2 RNAi (data not shown). (B) RNAi depletion of the downstream IIS kinases causes
sek-1-dependent accumulation of SKN-1 in intestinal nuclei. Expression of SKN-1B/C::GFP was monitored. (C) SKN-1C is phosphorylated by AKT-1, -2, and SGK-1 comparably to DAF-16, dependent upon
daf-2. Here and in (D)-(G), AKT-1::GFP, AKT-2::GFP, and SGK1::GFP were immunoprecipitated from WT or
daf-2(
e1370) C. elegans lysates with anti-GFP antibody, quantitated by western blotting with anti-GFP, and used to phosphorylate purified GST-fused SKN-1 or DAF-16 proteins with [gP32]ATP. GST controls are shown in Figure S4B. These kinases appropriately required activation in C. elegans, as they did not phosphorylate SKN-1 after they had been expressed in E. coli (Figure S4A). (D) The unique 90 aa region at the SKN-A N terminus (SKN-1A(1-90)) is phosphorylated by the AKT kinases but not SGK-1, dependent upon
daf-2. (E) Partial mapping of AKT-1 sites within SKN-1A(1-90). (F) AKT and SGK phosphorylate particular regions of SKN-1C. Here and in (G), residues are numbered as in SKN-1A, so that the first residue of SKN-1C is numbered as 91. (G) Partial mapping of AKT-1, -2, and SGK-1 sites within SKN-1C fragments. (H and I) Substitution of a strongly predicted AKT site within SKN-1A(1-90), Ser12 (Table S1), results in nuclear accumulation of SKN-1A::GFP. In some lines low levels of SKN-1::GFP were observed in additional head neurons, hypodermis, and other tissues, particularly if Ser12 was mutated (data not shown). p values were derived from a
chi2 test.