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LKB1 and its downstream kinase SIK3 are required for lipid homeostasis.

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posted on 21.05.2015, 02:55 authored by Sekyu Choi, Dae-Sik Lim, Jongkyeong Chung

(A) qPCR analysis of LKB1 and its cofactors required for the catalytic activity, STRAD and MO25, in Drosophila larvae under feeding condition. (B) TAG amounts of wild-type and LKB1 mutant larvae (n = 10 per genotype). (C) qPCR analysis for lipogenic genes (SREBP, FAS and ACC) and lipolytic genes (bmm and HSL) in wild-type and LKB1 mutant larvae at mid-to-late L2 (60 hr AEL) stage under feeding conditions. (D) qPCR analysis of LKB1, SIKs (SIK2 and SIK3), and AMPK complex (AMPKα, AMPKβ, and AMPKγ) in larvae. (E) TAG amounts in LKB1 mutants following fat body-specific expression of wild-type, kinase-dead (K201I) LKB1, constitutively active (T196E) SIK3 or constitutively active (T184D) AMPK. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), LKB1X5,FB>LKB1KI (FB-Gal4/UAS-LKB1 K201I;LKB1X5/LKB1X5), LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5), and LKB1X5,FB>AMPKTD (FB-Gal4/UAS-AMPK T184D;LKB1X5/LKB1X5) (n = 10 per genotype). (F) qPCR analysis of bmm gene expression in LKB1 mutants following fat body-specific expression of wild-type LKB1 or constitutively active (T196E) SIK3 at mid-to-late L2 stage under feeding condition. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), and LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5). (G) Immunoblot analyses showing the effect of LKB1 on Thr196 phosphorylation of SIK3 protein in larvae. Wild-type and kinase-dead (K70M) SIK3 were highly phosphorylated at Thr196 by LKB1 (second panel). SIK3T196A was used as a control. FB-Gal4 was used to drive transgene expression in the fat body. (H) Immunoblot analyses showing relative amounts of SIK3 Thr196 phosphorylation in wild-type and LKB1X5 mutant larvae. The phosphorylation was absolutely dependent on LKB1 (first panel). FB-Gal4 was used to drive transgene expression. (G-H) Anti-LKB1, -phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Data are presented as mean ± SEM (*P < 0.05).

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