Volume 13, Issue 10, Pages 861-866 (May 2003) A Novel Domain in AMP-Activated Protein Kinase Causes Glycogen Storage Bodies Similar to Those Seen in Hereditary Cardiac Arrhythmias  Emma R. Hudson, David A. Pan, John James, John M. Lucocq, Simon A. Hawley, Kevin A. Green, Otto Baba, Tatsuo Terashima, D.Grahame Hardie  Current Biology  Volume 13, Issue 10, Pages 861-866 (May 2003) DOI: 10.1016/S0960-9822(03)00249-5

Figure 1 Domain Organization of AMPK and the Effect of β Subunit Truncations on Activity (A) Domain map of the β1 subunit of AMPK and of the truncated mutants used in this study. The N-isoamylase/glycogen binding domain (GBD) and the KIS and ASC domains [9] are indicated. (B) Activities of the full-length and truncated β1 mutants when coexpressed with myc-α1 and -γ1 in CCL13 cells. Proteins were immunoprecipitated with anti-myc antibody, and kinase activity was measured in the precipitate. Activities are expressed relative to the activity obtained with the full-length β1 subunit (0.10 ± 0.01 nmol/min/mg lysate protein, mean ± SEM, n = 3) expressed at the same time. Current Biology 2003 13, 861-866DOI: (10.1016/S0960-9822(03)00249-5)

Figure 2 Truncated β Subunits Lacking the GBD Still Form Heterotrimeric Complexes (A) 35S-labeled CCL13 cells expressing myc-tagged α1, γ1, and full-length or truncated β1 were lysed and immunoprecipitated with anti-myc antibody; half the precipitate was analyzed by SDS-PAGE, blotting, and autoradiography. (B–D) The remaining half was analyzed in the same way, except that polypeptides were detected by (B) anti-α1, (C) anti-β1, or (D) anti-γ1 antibodies. The β1 construct transfected is indicated at the top in (A); migration of marker proteins is shown on the right, and identification of the polypeptides is shown on the left. The method used in (A) detects endogenous c-myc, which was also visible in untransfected cells (not shown). In (C), the secondary antibody also detects the light chain of sheep immunoglobulin (Ig light chain) used for immunoprecipitation. Current Biology 2003 13, 861-866DOI: (10.1016/S0960-9822(03)00249-5)

Figure 3 Cytoplasmic Inclusions in Cells Expressing GFP-Tagged AMPK Heterotrimers Contain Glycogen (A–F) Whole-cell projections of CCL13 cells expressing GFP-α1 and -γ1 with (A–C) β1 or (D–F) β2. Cells were fixed and stained with an anti-glycogen antibody that was detected with a Texas red-labeled secondary antibody. The pictures display the green channel (GFP; [A] and [D]), the red channel (Texas red; [B] and [E]), or both channels merged (C and F). The insets at the top-left of each panel show the largest cytoplasmic inclusion in each cell at higher magnification. The scale bars are in μm. Current Biology 2003 13, 861-866DOI: (10.1016/S0960-9822(03)00249-5)

Figure 4 Cytoplasmic Inclusions in Cells Expressing GFP-Tagged AMPK Heterotrimers Contain Glycogen Synthase (A–F) Whole-cell projections of CCL13 cells expressing GFP-α1, (A–C) β1, or (D–F) β2 and γ1. Cells were fixed and stained with an anti-glycogen synthase antibody that was detected with a Texas red-labeled secondary antibody. The pictures display the green channel (GFP; [A] and [D]), the red channel (Texas red; [B] and [E]), or both channels merged (C and F). DNA was stained with 4′,6-diamidino-2-phenylindole. The scale bars are in μm. Current Biology 2003 13, 861-866DOI: (10.1016/S0960-9822(03)00249-5)