Characterization of a new liver- and kidney-specific pfkfb3 isozyme that is downregulated by cell proliferation and dedifferentiation Joan Duran a , Marta Go ´mez a , Aurea Navarro-Sabate a , Lluı ´s Riera-Sans b , Merce ` Obach a , Anna Manzano a , Jose C. Perales c , Ramon Bartrons a, * a Unitat Bioquı ´mica i Biologia Molecular, Departament de Cie `ncies Fisiolo ` giques, Campus de Cie `ncies de la Salut, IDIBELL – Universitat de Barcelona, Spain b Laboratory of Epithelial Homeostasis and Cancer, Department of Differentiation and Cancer, Center for Genomic Regulation, Spain c Unitat de Biofı ´sica, Departament de Cie `ncies Fisiolo ` giques, Campus de Cie `ncies de la Salut, IDIBELL – Universitat de Barcelona, Spain Received 20 December 2007 Available online 10 January 2008 Abstract The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P 2 ), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1–4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cul- tured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiq- uitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism. Ó 2008 Elsevier Inc. All rights reserved. Keywords: 6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase; Fructose 2,6-bisphosphate; Hepatocytes; Alternative splicing; Isoform; Isozyme; Dedifferentiation; Proliferation Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) was discovered in liver during research into the mechanism of glucagon- stimulated gluconeogenesis [1,2]. This metabolite is the most powerful allosteric activator of 6-phosphofructo-1- kinase, as well as being an inhibitor of fructose-1,6-bis- phosphatase. Because of its antagonistic actions on these enzymes, Fru-2,6-P 2 plays a crucial role in governing flux along glycolytic and gluconeogenic pathways [1–3]. The Fru-2,6-P 2 cellular pull depends on the balance between the reciprocal reactions of 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase from the bifunctional enzyme PFK-2, which functions as a homodimeric protein with these two activities located at separate sites on each protein subunit [1–3]. Different PFK-2 isozymes have been identi- fied in mammals and are generated by alternative splicing of the transcribed RNA from four genes, designated Pfkfb1–4 [4]. PFK-2 isozymes differ in their kinetic proper- ties, molecular masses, and responses to phosphorylation by protein kinases, with the kinase:bisphosphatase activity ratio being the most important aspect of any given isozyme [5]. In the adult liver the main isozyme is L-PFK-2, a prod- uct of the Pfkfb1 gene that is modulated by protein kinase A phosphorylation, which activates its bisphosphatase activity that breaks down Fru-2,6-P 2 [5]. In embryonic or proliferating cells, studies have observed the expression of the Pfkfb3 gene [6–9]. This gene was originally cloned from a foetal bovine brain cDNA library [10,11], from placenta [12] and from a tumour cell line [13]. It has the highest 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.005 * Corresponding author. Fax: +34 934024268. E-mail address: rbartrons@ub.edu (R. Bartrons). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 367 (2008) 748–754