The human PFKFB3 is composed of 19 exons spanning genomic region about 90,6 Kb (GenBank). Alternative splicing variants have been reported. The main variants corresponding to mRNAs of 4453 bp and 4224 bp for the variant 1 u-PFK2 (NM_004566.3) and variant 2 i-PFK2 (NM_001145443.1), respectively (Figure 2).

The human PFKFB3 coding sequence consists of 1503 bp for i-PFK2 and 1563 bp for u-PFK2 from the start codon to the stop codon. Multiple alternatively spliced transcript variants have been found for this gene. (Entrez Gene) (OTTMUSG00000011314).

No pseudogene of PFKFB3 known.

PFKFB3 protein consists of two homodimeric subunits of 520 amino acids, with a molecular weight of 59609 Da for each subunit. The monomer structure is divided into two functional domains within the same polypeptidic chain (El-Maghrabi et al., 1982; Okar et al., 2001; Pilkis et al., 1995). The finding of mutations that affect kinase activity of the enzyme (Kurland et al., 1995; Li et al., 1992b; Rider et al., 1994), and the localization of ATPγS in the structure, lead to the identification of the N-terminal domain as the kinase domain, which catalyses the synthesis of Fru-2,6-P2, using fructose-6-phosphate and adenosine-5-triphosphate (ATP) as substrates (Figure 3). On the other hand, structural and sequence homology with yeast phosphoglycerate mutase (Winn et al., 1981) and rat acid phosphatase (Schneider et al., 1993), along with the location of mutations that affect Fru-2,6-Pase activity (Li et al., 1992a; Li et al., 1992c; Lin et al., 1992; Tauler et al., 1990) suggested the C-terminal domain to contain the bisphosphatase activity of the enzyme. This domain is responsible for the hydrolytic degradation of Fru-2,6-P2 into fructose-6-P and inorganic phosphate (Pi). These two mutually opposing catalytic activities are controlled by different mechanisms such that either activity is predominant in a given physiological condition. Ultimately, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) activities control Fru-2,6-P2 synthesis and degradation, regulating the rate of glucose metabolism.
Different PFK-2/FBPase-2 isoenzymes have been described, sharing high sequence similarity (85%). PFKFB3 isoenzyme has some structural differences with the other isoforms. The conformations of the substrate loops in the kinase domain are different from those of other isoforms (Hasemann et al., 1996; Lee et al., 2003), giving a structural explanation for the higher kinase activity. Moreover, the N-terminus binds to the bisphosphatase domain to produce a conformational change in the active pocket to enhance inhibitory binding of product (Kim et al., 2006). Residues 4-15 of the kinase domain form a β-hairpin structure and the rest is used as an arm connecting the hairpin to the bisphosphatase domain. Additionally, the contacting area of the bisphosphatase domain is functionally very sensitive due to the residues critical for binding of both product and substrate are located very close (Kim et al., 2006). The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms. This residue is said to interact with the 2-phosphate and further stabilizes the transitions state (Cavalier et al., 2012; Kim et al., 2006).

The PFKFB3 gene product is present in proliferating tissues (Duran et al., 2008a; Duran et al., 2009; Goren et al., 2000; Manzano et al., 1998; Sakai et al., 1996) and transformed cells (Calvo et al., 2006; Chesney et al., 1999; Hamilton et al., 1997; Minchenko et al., 2002; Novellasdemunt et al., 2012; Obach et al., 2004; Riera et al., 2002) and various tumors (Atsumi et al., 2002; Fleischer et al., 2011; Kessler et al., 2008). The high kinase/ bisphosphatase activity ratio of this isoenzyme can explain the high Fru-2,6-P2 found in the cells where it is present, which in turn sustains high glycolytic rates and it is crucial in supporting growing and proliferant cell metabolism.
PFKFB3 gene has been found to be expressed in different cell systems. During C2C12 myogenic cell differentiation, the 6-phosphofructo-2-kinase isoenzyme, product of the PFKFB3 gene, is downregulated, being the PFKFB3 isoenzyme degraded through the ubiquitin-proteasome proteolytic pathway (Riera et al., 2003). In neurons, PFKFB3 is constantly subjected to proteasome degradation after ubiquitylation by the E3 ubiquitin ligase APC/C-Cdh1. The activity of this complex is determinant in controlling the protein levels of PFKFB3 and hence the rate of glucose consumption through glycolysis. The function of APC/C is strictly dependent on the presence of Cdh1, which is very abundant in these cells. In contrast, astrocytes express very low Cdh1 levels and therefore APC/C activity is negligible. Accordingly, PFKFB3 protein levels are high and glycolysis is active in astrocytes owing to the low levels of Cdh1. In fact, overexpression of Cdh1 in astrocytes destabilizes PFKFB3 and concomitantly decreases the rate of glycolysis (Fernandez-Fernandez et al., 2012). The later finding that the activity of the PFKFB3 isoenzyme is regulated in cancer cells by APC/C-Cdh1 (Almeida et al., 2010) led to the identification of the role of this ubiquitin ligase in the metabolic regulation of the cell cycle and therefore of cell proliferation (Moncada et al., 2012).
The regulatory component of this complex, Cdh1, has been shown to be downregulated during malignant progression and tumor formation. A decrease in the activity of APC/C-Cdh1 in mid-to-late G1 phase, that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G1 to S, leads to the accumulation of PFKFB3 and enhances the glycolytic flux in malignant cells. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-β-TrCP (β-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G1, demonstrating that proliferation and the induction of aerobic glycolysis are both essential components of neoplastic transformation (Moncada et al., 2012).
PFKFB3 has also been found in testis. In adult testes both PFKFB3 and PFKFB4 isoenzymes are present. PFKFB3 is located across the seminiferous epithelium, whereas expression of PFKFB4 is restricted to the spermatogenic cells, being the only one present in mature spermatozoa (Gomez et al., 2005). This differential distribution supports the idea that the cell-specific isoenzymes are able to adapt their kinetic and regulatory enzymatic properties to the metabolic demand of a particular tissue or cell status. Thus, in parallel with spermatogenesis and spermiogenesis, PFKFB isoenzyme expression switches from PFKFB3, which is required during the proliferative phase, to the testis isoform PFKFB4, which is germ cell specific (Gomez et al., 2012; Gomez et al., 2009).
In mouse hypothalami, PFKFB3 mRNA levels are increased by 10-fold in response to re-feeding. In the hypothalamus, re-feeding also decreases the phosphorylation of AMP-activated protein kinase (AMPK) (Thr172) and the mRNA levels of agouti-related protein (AgRP), and increases the mRNA levels of cocaine-amphetamine-related transcript (CART). In addition, knockdown of PFKFB3 in N-43/5 neurons causes a decrease in rates of glycolysis, which is accompanied by increased AMPK phosphorylation, increased AgRP mRNA levels and decreased CART mRNA levels. In contrast, overexpression of PFKFB3 in N-43/5 neurons causes an increase in glycolysis, which was accompanied by decreased AMPK phosphorylation and decreased AgRP mRNA levels and increased CART mRNA levels. Together, these results suggest that PFKFB3 responds to re-feeding, which in turn stimulates hypothalamic glycolysis and decreases hypothalamic AMPK phosphorylation and alters neuropeptide expression in a pattern that is associated with suppression of food intake (Li et al., 2012).
Recent results have shown that the tumour suppressor PTEN promotes APC/C-Cdh1 activity (Song et al., 2011) and that cells from mice overexpressing PTEN exhibit reduced glucose and glutamine uptake and are resistant to oncogenic transformation (Garcia-Cao et al., 2012). Studies on the possible connection between APC/C-Cdh1 and the metabolic effects of other tumour suppressors such as p53 (Feng and Levine, 2010) or known proto-oncogenes such as c-Myc (Morrish et al., 2008) and Akt (Matheny and Adamo, 2009) are also likely to be highly relevant.

PFKFB3 protein is predominantly cytoplasmic but it has been found also into the nucleus. PFKFB3 overexpression produces a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-P2 which produced an increase in the expression of several key cell cycle proteins, including cyclin-dependent kinase Cdk1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle, indicating that Fru-2,6-P2 may couple the activation of glucose metabolism with cell proliferation (Yalcin et al., 2009a).

Synthesis and degradation of fructose-2,6-bisphosphate:
- Kinase catalytic activity: ATP + D-fructose-6-phosphate = ADP + beta-D-fructose-2,6-bisphosphate
- Phosphatase catalytic activity: Beta-D-fructose-2,6-bisphosphate + H2O = D-fructose-6-phosphate + phosphate
The rate of glycolytic flux is controlled at different levels and by different mechanisms: substrate availability, enzyme concentrations, allosteric effectors and covalent modification on regulatory enzymes. One of the critically modulated steps is that catalysed by 6-phosphofructo-1-kinase (PFK-1), with fructose-2,6-bisphosphate (Fru-2,6-P2) being its most powerful allosteric activator (Okar and Lange, 1999; Rider et al., 2004; Van Schaftingen, 1987). Fru-2,6-P2 relieves ATP inhibition and acts synergistically with AMP, and in addition it inhibits fructose 1,6-bisphosphatase (Van Schaftingen, 1987). These properties confer to this metabolite a key role in the control of fructose-6-P/fructose-1,6-P2 substrate cycle and hence critically regulate carbohydrate metabolism (Figure 4).

PFKFB genes
In mammals, there are four PFKFB genes (PFKFB1, PFKFB2, PFKFB3 and PFKFB4) which code for the different PFK-2/FBPase-2 isoenzymes, characterized by their cellular expression patterns. These isoforms share highly conserved core catalytic domains (85%) but differ greatly in their kinetic properties and responses to regulatory signals (Okar et al., 2001). These differences are mostly due to highly divergent N- and C-terminal regulatory domains; however, a few but significant sequence differences in the catalytic domains that constitute the secondary residue shells surrounding the active sites also contribute to the kinetic differences (Cavalier et al., 2012).
These isoforms show differences in their distribution and kinetic properties in response to allosteric effectors, hormonal, and growth factor signals (Okar et al., 2001). The expression of these genes is dependent on tissue and on development stage (Goren et al., 2000). Importantly, tissue- and cell-specific isoenzymes are not totally exclusive and several cells express more than one isoenzyme (Calvo et al., 2006; Minchenko et al., 2005a; Minchenko et al., 2005b; Telang et al., 2006). This pattern of expression suggests that each isoenzyme plays a key role under different physiological conditions or in response to different stimuli. Although the PFKFB isoenzymes have the same enzymatic activities and share the same substrates, indicating functional redundancy, their biological function and regulation is different in the specific cells (Table I).
PFKFB1 is mainly expressed in liver and skeletal muscle, PFKFB2 in heart tissue, PFKFB3 is expressed ubiquitously in several tissue and proliferating cells, and PFKFB4 was originally found in testis (Okar et al., 2001).
PFKFB3 has a uniquely large 6-phosphofructo-2-kinase to fructose-2,6-bisphosphatase activity ratio compared to other isoforms (Sakakibara et al., 1997). This isoform, which has a native activity ratio of roughly 700-fold kinase-to-phosphatase activity, dramatically increases upon phosphorylation of Ser461 by protein kinase A (PKA), AMP-dependent protein kinase (AMPK) or other kinases. The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is solely due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms (Cavalier et al., 2012; Kim et al., 2006). PFKFB3 gene was cloned from a fetal brain library (Manzano et al., 1998; Ventura et al., 1995), human placenta (Sakai et al., 1996) and breast cancer cells (Hamilton et al., 1997).
PFKFB3 is expressed ubiquitously and it is present in proliferating tissues, transformed cells and in tumours (Almeida et al., 2010; Atsumi et al., 2002; Bando et al., 2005; Calvo et al., 2006; Chesney et al., 1999; Duran et al., 2008a; Duran et al., 2009; Kessler et al., 2008; Novellasdemunt et al., 2012; Riera et al., 2002; Yalcin et al., 2009b). An inducible PFK-2/FBPase-2 (iPFK-2) with proto-oncogenic features was cloned from cancer cell lines (Chesney et al., 1999). The iPFK-2 represents a splice product of the PFKFB3 gene, as does the ubiquitous PFK-2/FBPase-2 (uPFK-2) (Navarro-Sabate et al., 2001). In human brain, have been demonstrated the occurrence of six alternatively spliced PFKFB3 transcripts, designated UBI2K1-6 splice isoforms of ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kessler and Eschrich, 2001).

Regulation:
PFKFB3 gene is regulated by different mechanisms. Induction of its expression has been reported in response to different stimuli, amongst these are hypoxia (Bartrons and Caro, 2007; Minchenko et al., 2002; Obach et al., 2004) and progestins (Hamilton et al., 1997; Novellasdemunt et al., 2012), through HIF (Hypoxia Inducible Factor) and PR (progesterone receptor) interactions within their binding to the consensus HRE (Hypoxia response element) and PRE (progesterone response element) sites located at PFKFB3 promoter, respectively. Growth factors such as insulin (Riera et al., 2002) and pro-inflammatory molecules (Chesney et al., 1999) such as IL-6 (Ando et al., 2010), LPS and adenosine (Ruiz-Garcia et al., 2011) or in response to stress stimuli (NaCl, H2O2, UV radiation or anisomycin) through SRF (Serum Response Factor) and its binding to SRE (Serum response element) site (Novellasdemunt et al., 2013) (Figure 5). The proinflammatory cytokine interleukin (Il-6) enhances glycolysis through activation of PFKFB3 as a consequence of the STAT3 activation (Ando et al., 2010). PFKFB3 is also a target gene of PPARγ. Additionally, PFKFB3 is involved in the antidiabetic effect of PPARγ activation, at least, by suppressing excessive fatty acid oxidation-related reactive oxygen species (ROS) production and inflammatory responses in adipose tissue/adipocytes (Huo et al., 2010).
The product of the PFKFB3 gene, the bifunctional enzyme 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase, is also controlled by different mechanisms. In addition to the gene expression regulation, the C-terminal domain can be phosphorylated at Ser461 by different protein kinases, such as AMP-activated protein kinase (AMPK) (Bando et al., 2005; Marsin et al., 2002), RSK (Novellasdemunt et al., 2012) and MK2 (Novellasdemunt et al., 2013) (Figure 6).
Ser461 can also be phosphorylated by PKC and PKA making it responsive to multiple external signals (Okamura and Sakakibara, 1998). Phosphorylated PFKFB3 kinetics shows an increase in Vmax of the kinase activity and a decreased Km for Fru-6-P (Marsin et al., 2002; Novellasdemunt et al., 2012). Furthermore, the PFKFB3 isoenzyme was found to be regulated through the PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) pathway, turning it into a target of growth factors signalling (Duran et al., 2009; Garcia-Cao et al., 2012) (Figure 7).
Furthermore, the mRNAs of all PFKFB3 isoforms contain multiple copies of the AUUUA instability motif in its 30 untranslated region (30UTR) (Chesney et al., 1999). AU-rich elements (AREs) target them RNAs of proto-oncogenes and pro-inflammatory cytokines for rapid degradation and regulate the efficiency of their translation into proteins (Chen and Shyu, 1995).
Also, PFKFB3 isoenzyme is regulated by modulation of its protein stability. Thus, it is degraded through the ubiquitin/proteasome proteolytic pathway (Riera et al., 2003). PFKFB3 but not the other isoenzymes, contains a recognition signal composed of a K-E-N box (KENXXXN), where K is lysine, E is glutamate and N is asparagine, that is recognized by the anaphase-promoting complex/cyclosome (APC/Cdh1), an E3 ubiquitin ligase complex that plays an essential role in G1 phase and mitosis through the degradation of several cell cycle proteins and PFKFB3 (Almeida et al., 2010).

It appears that the use of Fru-2,6-P2 as a regulatory metabolite is a specifically eukaryotic phenomenon. The most plausible hypothesis for the origin of the PFK-2/FBPase-2 would be the fusion of two ancestral genes coding for a kinase functional unit and a phosphohydrolase/mutase unit, respectively. From protein sequence alignments, it is clear that the bisphosphatase activity located in the C-terminal domain of the PFK-2/FBPase-2 is homologous to the phosphoglyceratemutases (PGMs) and the acid phosphatase family (Jedrzejas, 2000; Okar et al., 2001). Alignments of the bisphosphatase domain with PGM and acid phosphatase can be accessed at InterPro.
The N-terminal PFK-2 domain is sequentially and structurally homologous to several nucleotide binding proteins, primarily that of adenylate kinase of E. coli.

No PFKFB mutations have been collected in the COSMIC database. A lot of SNPs have been described, but they are not affecting to coding sequences. 2319 NCBI SNPs in PFKFB3 are shown (GeneCard).

No germinal mutations of PFKFB3 have been described.

Loss of heterozygosity (LOH) of PFKFB3 has been found in 55% of glioblastomas. The allelic deletion of PFKFB3 resulted in a decrease of PFKFB3 mRNA level accompanied by a lower PFKFB3 protein level. The expression of growth-inhibiting splice variant UBI2K4 was effectively reduced in glioblastomas with PFKFB3 LOH and a positive correlation with overall PFKFB3 expression was observed. The PFKFB3 LOH as well as the resulting low UBI2K4 expression level was associated with a poor prognosis of glioblastoma patients. It was concluded that LOH on 10p14-p15 in glioblastomas targets PFKFB3 and in particular splice variant UBI2K4, a putative tumor suppressor protein in glioblastomas (Fleischer et al., 2011).