PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4)

Description	PFKFB4 consists of 469 residues and has a molecular weight of 54040 Da. PFKFB4 is one of the four isoforms (PFKFB1-PFKFB2-PFKFB3-PFKFB4) of the bifunctional enzyme PFK2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), differing in their kinetic and regulatory properties. The PFK2 enzyme has both kinase and phosphatase activities, producing or degrading fructose-2,6-bisphosphate (Fru-2,6-P2). 6-Phosphofructo-2-kinase (6-PF-2-K) synthesizes Fru-2,6-P2 from fructose-6-phosphate (F6P) and ATP, while fructose-2,6-bisphosphatase (Fru-2,6-P2ase) hydrolyzes Fru-2,6-P2 to form F6P and inorganic phosphate (Pi). Fru-2,6-P2 is a rate-limiting enzyme product and an important control point in glycolysis and gluconeogenesis via its stimulatory effect on phosphofructokinase 1 (PFK1) activity and its inhibitory effect on fructose-1,6-bisphosphatase (Fru-1,6-P2ase) (Pilkis et al., 1996). The mammalian PFKFB4 gene encodes an isozyme originally identified as a homodimer in the testis (Sakata et al., 1991; Manzano et al., 1999; Gomez et al., 2005). The enzyme is divided into two functional domains (Figure 1): the N-terminal catalytic domain in which the 6-PF-2-K activity is found, and the C-terminal domain that houses the Fru-2,6-P2ase activity.
	Figure 1: Schematic representation of the structure of the bifunctional isozyme PFKFB4. The different potential sites were defined by mutagenesis or by structural similarity to other mononucleotide binding proteins and phosphoglucomutases (Hasemann et al., 1996; Yuen et al., 1999a; Yuen et al., 1999b; Tominaga et al., 1993; Uyeda et al., 1997).
Expression	PFKFB4 is expressed in testis and at specific times during sperm maturation (Gomez et al., 2009). A recent report showed that 5α-androstan-17β-ol-3-one, inducing the paracrine secretion of FGF-2 by Sertoli cells could modulate the expression of PFKFB isozymes during spermatogenesis (Gomez et al., 2012). Moreover, it was demonstrated that hypoxia, as well as glucose level, strongly regulate PFKFB4 mRNA and protein expression levels in different cancer cell lines from prostate and liver (Minchenko et al., 2004; Li et al., 2012; Ros et al., 2012). Recent studies have showed the cancer-specific overexpression of PFKFB4 in astrocytoma (Goidts et al., 2012).
Localisation	PFKFB4 is found in the cytosol.
Function	PFKFB4 is a bifunctional isozyme harboring two domains that function within a homodimeric protein complex. It synthesizes and degrades Fru-2,6-P2 as described in the equations above (Figure 2) (Sakata et al., 1991). The first reaction (kinase) taking place at the N-terminal side of the protein transfers the phosphate from ATP to F-6-P by a sequential-ordered mechanism. The biphosphatase reaction, which is located in the C-terminal domain, proceeds via a covalent phosphohistidine intermediate at position H257 (Figure 1) formed upon reaction with F-2,6-P2. The steady state concentration of F-2,6-P2 is determined by the balance between these opposing reactions. The kinase:biphosphatase activity ratio is different for all four isoforms (Okar et al., 2001). Indeed, the K:B ratio of the human PFKFB4 is approximately of 1 while PFKFB3 harbors a significantly higher ratio (740:1). These differences mainly reside on the effect of several glycolytic metabolites or post-translational modifications. Fru-2,6-P2 is a powerful allosteric activator of phosphofructokinase 1 (PFK-1), the enzyme that controls one of the most critical steps of glycolysis (Wu et al., 2006). However, Fru-2,6-P2 not only controls the PFK-1 reaction but also the reverse reaction in the gluconeogenic pathway by inhibiting fructose 1,6-bisphosphatase (FBPase-1) (Figure 3).
	Figure 3: Overview of the function of PFK2. 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases (PFK-2/FBPase-2) regulates the production of F-2,6-P2 which controls the production of F-1,6-P2 by activating or inhibiting PFK1.
Homology	There are four PFK2 isoenzymes in mammals encoded by four different genes (PFKFB1-PFKFB4). Although the different isoforms present a high sequence homology of their core domain, there are differences in their regulatory and kinetic properties, probably due to the structural variations in the N- and C-terminal regions (Ros and Schulze, 2013). From protein sequence alignments, it appears that the bisphosphatase activity is homologous to the phosphoglycerate mutases (PGMs) and the acid phosphatase family. The N-terminal PFK-2 domain is homologous to several nucleotide binding proteins, such as the NMP kinase (nucleoside-monophosphate kinase) (Bazan et al., 1989; Hasemann et al., 1996; Okar et al., 2001).

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