Fig. 1

AMPK phosphorylates mouse TET2 at S97 in vitro and in vivo. a Murine TET2 harbors a well-established substrate motif of AMP-activated protein kinase (AMPK) around Ser 97. The logo motif of AMPK phosphorylation sites was generated by Web Logo [73] using data curated by Hardie et al. [14]. The residues surrounding S97 of murine TET2 which AMPK prefers are shown in red. The AMPK target motif around murine (S97) of TET2 is conserved across different species. b Phosphorylation of TET2 at Ser97 was detected in the product of an in vitro AMPK kinase reaction by mass spectrometry. c A phosphor-specific antibody against pSer97 of TET2 recognized wild-type TET2 phosphorylated by AMPK in vitro, but not a Ser97Ala (S97A) mutant. d Treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) induced TET2 phosphorylation. HEK293T cells transiently transfected with FLAG-tagged TET2 were treated with 2 mM AICAR for 24 h. FLAG-TET2 was immunoprecipitated with M2 beads (Sigma), and phosphorylation of TET2 was detected by Western blot analysis. e No glucose or 2-deoxy-d-glucose (2-DG) induced FLAG-TET2 phosphorylation. HEK293T cells were transiently transfected with FLAG-tagged TET2; cells were starved of glucose for 24 h or treated with 25 mM 2-DG for 2 h. FLAG-TET2 was immunoprecipitated with M2 beads (Sigma), and phosphorylation of TET2 was detected by Western blot analysis. f HEK293T cells were transiently transfected with FLAG-TET2 (WT or S97A) and GST-AMPKa1 (residues 1-312) for 24 h. FLAG-TET2 was immunoprecipitated with M2 beads (Sigma), and phosphorylation of TET2 was detected by Western blot analysis. g Knockout of AMPK diminished the phosphorylation of TET2 at Ser97. Expression of total TET2 and phosphor-TET2 (Ser97) in wild-type (WT) or AMPK knockout (KO) mouse embryonic fibroblasts (MEFs) cells was detected by Western blot analysis. h Phosphorylation of TET2 does not affect the interaction between TET2 and O-GlcNAc transferase (OGT). Immunoprecipitation of TET2 in wild-type (WT) or AMPK knockout (KO) mouse embryonic fibroblasts (MEFs) cells was followed by Western blot analysis using indicated antibodies. *a non-specific band