Journal: The Journal of Cell Biology
Article Title: GLP-1 signaling suppresses menin’s transcriptional block by phosphorylation in β cells
doi: 10.1083/jcb.201805049
Figure Lengend Snippet: Ex-4 induces phosphorylation of menin at the Ser487 residue through PKA. (A) Purified recombinant His-menin proteins were incubated with active PKA Cα in the presence of ATP. After SDS-PAGE, the phosphorylation was tested with anti-phospho-(Ser/Thr) PKA substrate antibody. Anti-menin antibody showed that equal amount of menin was used for the kinase assay. (B) After overnight starvation, INS-1 cells were treated with 10 nM Ex-4, 10 µM forskolin, 100 µM dibutyryl-cAMP, or 10 µM 8-pCPT-2′-O-Me-cAMP-AM for 30 min and then lysed for immunoprecipitation (IP) with anti-menin antibody. Proteins retained on sepharose were blotted with the indicated antibodies. (C) 2 h before forskolin or Ex-4 treatment, 2 µM Rp-cAMPS was added to the starvation medium, and the same experiment described in B was conducted. (D) The Ser487 residue of menin is highly conserved among species. (E) HEK293 cells were transfected with full-length Flag-menin WT, S122A, or S487A mutant, followed by IP using anti-Flag antibody. Proteins retained on sepharose were incubated with active PKA Cα for the kinase assay and then blotted with anti-phospho-(Ser/Thr) PKA substrate antibody. (F) Purified His-menin proteins (WT, S122A, or S487A mutant) were incubated with active PKA Cα in the presence of ATP. After SDS-PAGE, the phosphorylation was tested with anti-phospho-(Ser/Thr) PKA substrate antibody. (G) HEK293 cells cotransfected with Flag-menin (WT or S487A or S487D mutant) and GFP-PKA Cα were lysed and subjected to Western blot using the indicated antibodies. (H) INS-1 cells, treated with 10 nM Ex-4, 10 µM forskolin for 30 min after overnight starvation, were lysed for IP with anti-menin antibody, followed by Western blot with the indicated antibodies. 2 µM Rp-cAMPS was added to the starvation medium 2 h before treatment, if applicable.
Article Snippet: The antibodies and chemicals used in this study were as follows: rabbit polyclonal anti-menin (A300-115A, 1:5,000 dilution; Bethyl), mouse monoclonal anti-Flag (clone M2; F3165, 1:1,000 dilution; Sigma-Aldrich), rabbit polyclonal anti-PKA Cα (4782, 1:1,000 dilution; Cell Signaling Technology), rabbit monoclonal anti-phospho-PKA substrate (RRXS*/T*; 9624, 1:1,000 dilution; Cell Signaling Technology), rabbit monoclonal anti-GFP (2956S, 1:1,000 dilution; Cell Signaling Technology), mouse monoclonal anti-insulin (clone L6B10; 8138, 1:1,000 dilution; Cell Signaling Technology), rabbit monoclonal anti-GAPDH (2118, 1:1,000 dilution; Cell Signaling Technology), rabbit polyclonal anti-Myosin IIa (3403, 1:1,000 dilution; Cell Signaling Technology), rabbit polyclonal anti-histone H3 (trimethyl K4) antibody (ab8580; Abcam), rabbit polyclonal anti-histone H3 (trimethyl K9) antibody (ab8898; Abcam), mouse monoclonal anti-histone H3 (trimethyl K27) antibody (ab6002; Abcam), rabbit polyclonal anti-histone H3 (ab1791; Abcam), mouse monoclonal anti-Suv39H1 (ab12405, 1:1,000 dilution; Abcam), rabbit polyclonal anti-HDAC1 (ab7028, 1:1,000 dilution; Abcam), rabbit polyclonal anti-IgG (ab171870; Abcam), rabbit polyclonal anti-lamin A (ab26300, 1:2,000 dilution; Abcam), rabbit polyclonal anti-XPO6 (ab72333, 1:1,000 dilution; Abcam), rabbit polyclonal anti-acetyl-histone H3 (06–599; Millipore), mouse monoclonal anti-β actin (SC47778, 1:1,000 dilution; Santa Cruz), mouse monoclonal anti-pSer487 menin (1:1,000 dilution; this paper), goat anti-mouse IgG (H+L) HRP conjugate (170–6516, 1:5,000 dilution; Bio-Rad), Goat Anti-Rabbit IgG (H+L) HRP conjugate (170–6515, 1:5,000 dilution; Bio-Rad), Clean-Blot IP detection kit (HRP; 21232, 1:400 dilution; Thermo Scientific), Alexa Fluor 488 goat anti-mouse IgG (H+L; A11001, 1:1,000 dilution; Invitrogen), Ex-4 (1269105; Sigma-Aldrich), forskolin (F6886; Sigma-Aldrich), ATP disodium salt hydrate (FLAAS; Sigma-Aldrich), PKA catalytic subunit from bovine heart (P2645; Sigma-Aldrich), Chaetocin from Chaetomium minutum (C9492; Sigma-Aldrich), trichostatin A (T1952; Sigma-Aldrich), SAHA (SML0061; Sigma-Aldrich), 8-pCPT-2′-O-Me-cAMP (sc-257020; Santa Cruz), dibutyryl-cAMP (sc-201567; Santa Cruz), Rp-cAMPS (sc-24010; Santa Cruz), leptomycin B (9676; Cell Signaling Technology), jasplakinolide (ab141409; Abcam), cytochalasin D (ab143484; Abcam), and latrunculin B (ab144291; Abcam).
Techniques: Purification, Recombinant, Incubation, SDS Page, Kinase Assay, Immunoprecipitation, Transfection, Mutagenesis, Western Blot
