Nuclear Compartmentalization of a Key Regulator of Glycolysis, 6‐Phosphofructo‐2‐Kinase (PFKFB3)

6‐Phosphofructokinase‐2/fructose‐2,6 bisphosphatases‐3 (PFKFB3) is a bifunctional enzyme that is responsible for both synthesis and degradation of fructose‐2,6‐bisphosphate (F2,6BP), a potent allosteric activator of 6‐phosphofructo‐1‐kinase (PFK‐1). There exist six alternative carboxy (C)‐terminal splice variants of PFKFB3. As determined by RT‐PCR analysis, the PFBFB3 C‐terminal splice variant 5 is most highly expressed in several tissues examined. We then studied the subcellular localization of the PFKFB3 splice variant 5 as well as three other splice variants that were expressed in various tissues (variants 3, 4 and 6). Surprisingly, immunofluorescence microscopy analyses revealed that the dominantly expressed splice variant 5 localized to the nucleus. We then found that two lysine residues at 472 and 473 are required for nuclear targeting of the PFKFB3 splice variant 5. F2,6BP analysis in cells transfected with wild‐type or double alanine mutant (K472A/K473A) PFKFFB3 (cytoplasmic) revealed that nuclear localization of PFKFB3 decreases F2,6BP production (WT nuclear PFKFB3, 7 +/‐ 6.1 pmol/mg protein; re‐routed cytoplasmic PFKFB3, 13 +/− 9 pmol/mg protein). In conclusion, these data indicate that PFKFB3 may be sequestered in the nucleus in order to compartmentally regulate the activity of PFKFB3 and thus glycolysis. This work was supported by National Institutes of Health COBRE Grant P20RR018733‐01