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. Author manuscript; available in PMC: 2012 May 13.
Published in final edited form as: Cell. 2011 May 13;145(4):596–606. doi: 10.1016/j.cell.2011.04.013

Figure 2. AKT stimulates SIK3 activity during feeding, also see Figure S2.

Figure 2

(A) Effect of fat-body specific AKT expression on lipid levels in wild-type and SIK3− flies. Genotypes are: FB> (FB-GAL4/+), FB>AKT (FB-GAL4/UAS-AKT), SIK3−, FB> (FB-GAL4,SIK348/SIK348) and SIK3−, FB>AKT (FB-GAL4,SIK348/UAS-AKT, SIK348). (n=16 per genotype).

(B) SIK3 catalytic activity in 24 hour fasted versus 0.5 hour refed flies, as determined by relative SIK3 autophosphorylation in fat bodies of flies (ppl-GAL4/UAS-SIK3.WT) expressing HA-tagged SIK3. Incorporation of γ32P-labeled ATP by in-vitro kinase assay of anti-HA immunoprecipitates shown. Immunoblot of total SIK3 protein amounts also indicated.

(C) Immunoblot showing relative amounts of phosphorylated SIK3 in fat bodies of wild-type and Chico mutant flies. Phospho-SIK3 levels determined using phospho-AKT substrate antibody (PAS). Total SIK3 levels measured with SIK3 antisera on HA-immunoprecipitates prepared from CON (ppl-GAL4,UAS-SIK3/+) and Chico (chico1;ppl-GAL4,UAS-SIK3/+) flies under 24 hour fasted or 0.5 hour refed conditions.

(D) In vitro kinase assays showing effect of recombinant AKT on phosphorylation of catalytically inactive (K70M) SIK3. Effect of mutating four putative AKT phosphorylation sites (T281/486A.S293/401A; 4A) in SIK3 on 32P incorporation shown. Total protein amounts for wild-type and 4A mutant SIK3 proteins indicated by Coomassie staining.

(E) Immunoblot showing effect of Ala substitutions at putative AKT phosphorylation sites in SIK3 (4A) on SIK3 phosphorylation in Drosophila S2 cells. Immunoblot with PAS antiserum was performed on HA-immunoprecipitates prepared from transfected cells. Exposure to insulin (0.5 hr) indicated. Total amounts of SIK3 shown. Relative amounts of phospho-SIK3, determined by densitometric analysis, indicated below each lane.

(F) Lipid levels in SIK3 mutant flies following fat body specific expression of wild-type or AKT phosphorylation-defective (4A) SIK3. Genotypes are: FB>(FB-GAL4/+), SIK3−, FB> (FB-GAL4, SIK348/SIK348), SIK3−, FB>SIK3.WT (FB-GAL4, SIK348/SIK348;UAS-SIK3/+) and SIK3−, FB>SIK3.4A (FB-GAL4, SIK348/SIK348;UAS-SIK3.4A/+). (n=16 per genotype). Data are averages ± SEM (**, P<0.01; *, P<0.05).