Figure 3.
The carboxy-terminal tail of AMPKα functions as an NES. (A) Schematic representation of the four constructs containing two tandem GFP molecules (2xGFP) with or without the carboxy-terminal 23 amino acids of AMPKα2 (red) and/or the SV40 NLS (blue) used in B. (B) HEK293 cells transfected with constructs containing 2xGFP alone, 2xGFP with the SV40-NLS, 2xGFP with the AMPKα2 tail, or 2xGFP with both the SV40 NLS and the α2 tail. Nuclei were stained with DRAQ5 (blue). (C) Alignment of the AMPKα2/AMPKα1 tail with confirmed CRM1-dependent NESs. The CRM1-NES consensus sequence is shown, consisting of either four or five bulky hydrophobic amino acids (ϕ, red) with variable spacing between them. Key leucine residues (often enriched in CRM1 NES sequences), L546 and L550, that were mutated for D are highlighted with asterisks (*). (D) Cells expressing either 2xGFP fused to the wild-type α2 tail and treated with the CRM1-specific inhibitor leptomycin B (LMB), or 2xGFP with L546A and L550A (L,L→A,A) mutations in the α2 tail, to block CRM1-mediated nuclear export (compare to α2 tail in B). (E) Cells transfected with a GFP-tagged full-length AMPKα2L,L→A,A mutant. (F) Scoring of cells for subcellular localization of GFP-AMPKα with the L,L→A,A mutations to nucleus (N), nucleus and cytoplasm (NC) or cytoplasm (C; as in Figure 2D). Bar, 10 μm.