(A) GO enrichment analysis showing that PFKP interacts with multiple proteins functioning in regulation of nuclear transcription (colored circles with highest protein counts). Source data from 123 literature-curated PFKP-interacting proteins were obtained from the BioGRID database. “Protein Counts” indicates the number of proteins interacting with PFKP in each functional annotation cluster. (B and C) PFKP nuclear localization relies on importins and exportins. (B) Immunoblotting (IB) shows decreased PFKP in nuclear extract (NE) of cells treated with the importin inhibitor, importazole (Impor) (40 μM, 24 hours). (C) Increased PFKP in the nuclei of cells treated with the exportin inhibitor, leptomycin B (LMB) (5 ng/mL, 24 hours). CE, cytoplasmic extract. (D and E) Nuclear-to-cytoplasmic ratios of GFP protein expression from DU145 cells expressing NES-GFP (D) or NLS-fused GFP (E). IB of GFP was quantified using ImageJ (NIH). GFP vector served as control. (F) Nuclear enrichment of PFKP NES mutant. (G) Reduced nuclear accumulation of PFKP NLS mutant. In F and G, IB was performed on the NEs and CEs of cells expressing FLAG-tagged PFKP (left) with an anti-FLAG antibody. The graph on the right shows the NE/CE ratios of FLAG-PFKP. Lamin A/C is a nuclear protein marker. Tubulin is a cytoplasmic protein marker. (H) Localization of functional NESs (NES1 in green, NES2 in blue, left) and NLS (in red, right) in the structure of the tetrameric PFKP protein. Functional NLS localizes at the interface in the dimeric form of PFKP. Pymol software was applied for protein structure analysis, and the structure of human PFKP (DOI: 10.2210/pdb4U1R/pdb) was downloaded from the Protein Data Bank (37). n = 3 (B–G). Data represent mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001 by 2-tailed Student’s t test (F and G) or 1-way ANOVA (D and E).