Figure 1.

SILAC studies identified SIK3 as a new mTORC1 substrate. (A) Murine brown adipose cell labeling and treatment for SILAC analysis. Isotope labeled cells that received rapamycin were pretreated for 30 min, and then treated with isoproterenol or insulin for 15 min. Cell lysates were processed and subjected to liquid chromatography and mass spectrometry analysis to profile the phosphorylated peptides. (B) Venn diagram illustration of the representative protein whose phosphorylation is increased by isoproterenol (Iso, blue) or insulin (Ins, orange) and blocked by rapamycin (Rapa, green). (C) The phosphorylation of S6 Ser²⁴⁰, AKT Ser⁴⁷³ and SIK3 Ser⁸⁸⁴ in response to Iso, Ins and Rapa treatment. Data are presented as log2 fold change compared to the vehicle treated samples. ∗p < 0.05. (D) SIK protein structure and phosphorylation sites. The LKB1 (Liver kinase B1, orange), PKA (blue), and mTORC1 (S884, red) phosphorylation sites are indicated. KD: kinase domain. UBA: ubiquitin-associated domain. (E) HEK293FT cells were transfected with SIK3-Flag and/or RAPTOR-Myc plasmids as indicated. Protein lysates were immunoprecipitated with anti-Flag beads and blotted with indicated antibodies.