The human PFKFB2 is composed of 15 exons spanning 22617 bp (GenBank: AJ005577.1). This gene has 9 transcripts; two of them have been reported to codify a protein and three contain an open reading frame, but no protein has been identified. The transcripts are derived from different promoters and vary only in non-coding sequences at the 5 end. Therefore, the resulting proteins differ in their C-terminal amino acid sequence (Heine-Suñer et al., 1998). The main products of the gene correspond to mRNAs of 7073 bp and 3529 bp for the variant 1 (isoform a; NM_006212.2) and variant 2 (isoform b; NM_001018053.1), respectively (Fig. 2). The isoform b differs in the 3 UTR and the coding region compared to isoform a. The resulting isoform b is shorter and has a distinct C-terminus compared to isoform a. However, it is not known how these different 5 ends are related to the three mRNAs (H1, H2 and H4) that encode the isoform a or the H3 mRNA that encodes the isoform b. None of these mRNAs are strictly heart-specific.
The overall gene structure of the human PFKFB2 gene has exons clustered into three groups. The first group contains exons 1 and 2 that are different from those in other PFKFB2 genes and contain the ATG initiation codon in exon 2. The second group contains exons 3-8 coding for the PFK-2 domain and the third group contains exons 8-15 coding for the FBPase-2 domain and a carboxy-terminal regulatory domain. Gene structure, exon-intron organization, as well as intron sizes, are similar to those of the rat and bovine homologous genes.

The human PFKFB2 coding sequence consists of 1518 bp for isoform a and 1416 bp for isoform b from the start codon to the stop codon, although the immature transcript forms contain 7904 bp and 3494 bp, respectively. Multiple alternatively spliced transcript variants have been described for this gene (Ensembl: OTTHUMG00000036033).

No pseudogene of PFKFB2 is known.

PFKFB2 is a homodimeric protein of 505 amino acids for isoform a and 471 for isoform b with a deduced molecular mass of 58 kDa and 54 kDa, respectively.
PFKFB2 is an enzyme of PFKFB family, as it shares different structure and function with the others isoenzymes. PFKFB2 has two distinct catalytic sites in each subunit: one for the 6-phosphofruto-2-kinase (PFK-2) activity and the other for the fructose-2,6-bisphosphatase (FBPase-2) activity (El-Maghrabi et al., 1982; Pilkis et al., 1995; Okar et al., 2001). The sequence of the catalytic core is highly conserved, whereas the N-terminal and C-terminal regions show more divergence (Rider et al., 2004).
PFK-2/FBPase-2 activities control fructose-2,6-bisphosphate (Fru-2,6-P2) synthesis and degradation, regulating the rate of glucose metabolism. More information about PFKFB2 protein can be found in Uniprot O60825.

PFKFB2 protein is expressed mainly in heart, although expression is also found in other tissues at lesser extent (Minchenko et al., 2002). Moreover, it is expressed in different cancer cell lines such as T-lymph Jurkat, K562 erythroleukemia, liver HepG2, lung A549, colon RKO, bone U2OS, brain GAMG, prostate LnCap, cervix HeLa and breast MCF7. All this information can be found in GeneCards (sections proteins and expression).
According to the RNAseq database, this gene can also be expressed in thyroid, brain, kidney, skeletal muscle, ovary, testis and others.

PFKFB2 protein is active in the cytosol.

This enzyme regulates the concentration of Fru-2,6-P2 through the two catalytic domains. PFK-2 domain catalyzes the synthesis of Fru-2,6-P2, using fructose-6-phosphate (Fru-6-P) and adenosine-5-triphosphate (ATP) as substrates; FBPase-2 domain catalyzes the degradation of Fru-2,6-P2 into Fru-6-P and inorganic phosphate (Pi). These two mutually opposing catalytic activities are controlled by different mechanisms such that each activity is predominant in a given physiological condition. In detail, the reactions catalyzed are:
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 modifications on regulatory enzymes. One of the critically modulated steps is that catalyzed by 6-phosphofructo-1-kinase (PFK-1), in which Fru-2,6-P2 is the most powerful allosteric activator (Van Schaftingen, 1987; Okar and Lange, 1999; Rider et al., 2004). Fru-2,6-P2 relieves ATP inhibition and acts synergistically with adenosine monophosphate (AMP), inhibiting fructose 1,6-bisphosphatase (Fru-1,6-Pase) (Van Schaftingen, 1987). These properties confer to this metabolite a key role in the control of Fru-6-P/Fru-1,6-P2 substrate cycle and hence critically regulates carbohydrate metabolism (Fig. 3).
In vertebrates, there are four different PFKFB genes (PFKFB1, PFKFB2, PFKFB3 and PFKFB4), which encode the PFK-2/FBPase-2 isoenzymes. Each of these enzymes has been originally identified in different mammalian tissues: PFKFB1 in liver and skeletal muscle, PFKFB2 in heart, PFKFB3 in brain, adipose tissue and proliferating cells, and PFKFB4 in testis (Okar et al., 2004; Rider et al., 2004). However, all four are widely expressed throughout the adult organism. These isoenzymes show differences in their distribution and kinetic properties in response to allosteric effectors, hormonal, and growth factors signals (Okar et al., 2001). PFKFB2 enzyme is overexpressed in different cancer cells like melanoma, prostate, pancreatic, gastric and mammary gland cells (Minchenko et al., 2005a; Minchenko et al., 2005b; Bobarykina et al., 2006). For more information about PFKFB genes see: PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) and PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4).

Regulation
PFKFB2 is an essential enzyme for the regulation of glycolysis in heart. PFKFB2 is multisite-phosphorylated, integrating signaling from many pathways via protein kinase cascades to a single molecule, Fru-2,6-P2, to stimulate glycolysis.
The human PFKFB2 protein contains the Ser 29, Ser 466, Thr 475 and Ser 483 residues that regulate the activity of the enzyme. These residues are located in its C-terminal domain and can be phosphorylated by protein kinases such as AMPK, 3-phosphoinositide-dependent kinase-1 (PDK-1), cAMP-dependent protein kinase (protein kinase A; PKA), protein kinase B (PKB; also known as Akt), p70 ribosomal S6 kinase (S6K1), and mitogen-activated protein kinase 1 (MAPK-1). Phosphorylation of PFKFB2 results in the activation of the enzyme, increasing Vmax of PFK-2 activity. The variations in PFK-2 activity, however, appear to be different with the phosphorylation by the different kinases (Marsin et al., 2000; Rider et al., 2004).
In perfused rat hearts, it has been shown that the concentration of Fru-2,6-P2 is raised by increasing the work load, after hypoxia or stimulation with adrenalin or insulin (Hue et al., 1982; Rider and Hue, 1984; Depre et al., 1993; Deprez et al., 1997). This activation is probably mediated by the phosphorylation of three conserved residues (Ser 466, Thr 475 and Ser 483) by specific protein kinases (Depre et al., 1993; Deprez et al., 1997).
Insulin stimulates glycolysis in heart by a combination of an increase in glucose transport and activation of PFKFB2 (Depré et al., 1998; Hue et al., 2002). Two serine residues, Ser 466 ad Ser 483 can be phosphorylated in vitro by PKB in response to insulin resulting from a 2-fold increase in both Vmax and affinity for Fru-6-P, one of the substrates of PFK-2 (Lefebvre et al., 1996; Deprez et al., 1997).
Rat heart PFKFB2 is activated by insulin in vivo through a 2-fold increase in Vmax with no change in Km for Fru-6-P (Rider and Hue, 1984). Moreover, it has been shown that the insulin-induced activation of PFKFB2 was blocked by wortmannin, a PI3K inhibitor, but was insensitive to rapamycin or PD098059, which prevent the activation of p70^S6K and the MAPK cascade, respectively (Lefebvre et al., 1996). These results suggest that PI3K, but not p70^S6K, is involved in the activation of PFKFB2 in response to insulin. New in vitro and in vivo experiments show that SGK3 is not required for insulin-induced heart PFK-2 activation and this effect is likely mediated by PKBα (Mouton et al., 2010). Moreover, it has been proposed that 14-3-3s, that have been implicated in promoting cell survival (Masters et al., 2002), bind to PFK-2 at Ser 483 when it is phosphorylated by PKB in vitro in response to insulin or in cells that are stimulated with IGF-1 or transfected with active forms of PKB, mediating the stimulation of glycolysis by growth factors (Pozuelo et al., 2003).
Glycolysis in heart also increases in response to increased the workload (Depre et al., 1993; Beauloye et al., 2002), rising Fru-2.6-P2 due to the activation of PFKFB2. The increase on workload activates PKB but not p70 ^S6K and this increase is blocked by wortmannin and is rapamycin-insensitive. Ca/CAMK (Ca²⁺/calmodulin-activated protein kinase) is which phosphorylates and activates PFKFB2 secondarily to a rise in cytoplasmatic Ca2²⁺ (Depre et al., 1993; Beauloye et al., 2002).
Adrenalin administration in perfused rat hearts suggests that PKA may be responsible for the activation of PFKFB2, which accounts for the increased Fru-2,6-P2 levels (Narabayashi et al.,1985). This hormone promotes PFKFB2 phosphorylation by PKA in the residues already described in vitro, which are Ser 466 and Ser 483. These phosphorylations have an impact on PFK-2 activity, decreasing the Km for Fru-6-P (Kitamura et al., 1988; Rider et al., 1992a; Rider et al., 1992b).
PFKFB2 mRNA is induced in organs exposed to hypoxic conditions. Activation of the AMP-activated protein kinase (AMPK) during ischemia or hypoxia leads to phosphorylation of PFKFB2 at Ser 466 which increases the levels of Fru-2,6-P2 and stimulates glycolysis. PFKFB2 phosphorylation leads to an increase in Vmax with no change in Km for Fru-6-P (Marsin et al., 2000). Other studies have described PFKFB2 as a hypoxia-responsive gene in vivo but the regulation of its expression following hypoxic treatments appears to occur in a cell-specific manner. The mechanism underlying the expression of each isoform in different tissues remains unclear (Minchenko et al., 2002).
Moreover, amino acids increase the synthesis of Fru-2,6-P2 in HeLa and MCF7 cell lines by phosphorylation at PFKFB2 at Ser 483. This activation is mediated by PI3K and PKB. Similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, PFKFB2 phosphorylation increases glucose consumption and the production of lactate and ATP (Novellasdemunt et al., 2013).
PFKFB2 is also a substrate of PKC which phosphorylates Ser 84, Ser 466 and Thr 475 (Rider and Hue, 1986; Kitamura et al., 1988; Rider et al., 1992a; Rider et al., 1992b). However, the physiological significance of phosphorylation of Ser 84, Ser 466 and Thr 475 of PFKFB2 by PKC is not completely understood. It seems that phosphorylation of Ser 466 or Thr 475 does not change the enzyme activity. This might be due to the fact that the phosphorylation at Ser 84 possibly counteracts the effects of phosphorylation at the activating C-terminal sites (Kitamura et al., 1988; Rider et al., 1992b).
The mechanism of control of PFKFB2 isoenzyme by phosphorylation is also difficult to explain in the absence of a crystal structure of the phosphorylated isoenzyme. Phosphorylation of Ser 466 and Ser 483 at the C-terminal end of the bovine heart isoenzyme by PKA (Kitamura et al., 1988; Rider et al., 1992a; Rider et al., 1992b; Deprez et al., 1997) and insulin-stimulated protein kinases (Deprez et al., 1997) activates PFK-2 by decreasing Km for Fru-6-P and by increasing the Vmax without affecting FBPase-2. Ser 466 phosphorylation is responsible for the increase in Vmax whereas both phosphorylations are necessary to decrease the Km for Fru-6-P (Bertrand et al., 1999).
Regulatory sequences that account for some of the mechanisms involved in the long-term hormonal control and tissue-specific expression of PFKFB2 have been identified. The 5 flanking sequence of PFKFB2 contains regions that are conserved between the human, bovine and rat genes. In these regions, several potential binding sites for the Sp1, HNF-1 and BHLH (helix-loop-helix) (E boxes) transcription factors and for the GR have been described (Tsuchiya and Uyeda, 1994; Chikri and Rousseau, 1995; Heine-Suñer et al., 1998), but a factor binding to these sites has not been reported.

Chromosomal rearrangements: copy number variants
There are three alterations affecting PFKFB2 genome region described in patients. One of them, the gain of 1:195266734-216326885, shows phenotypic effects such us visual impairment, low-set ears, iris coloboma, intellectual disability, defect in the atrial septum, ventricular septal defect and vertical nystagmus. For more information see DECIPHER.
No syndrome or disease was found in OMIM database.

Location in the mouse: chromosome 1, 56,89 cM, cytoband E4, 130689043-130729253 bp, complement strand (MGI).
For a comparison of the gene from Homo sapiens, mouse, rat, cattle, chimpanzee, chicken, zebrafish, rhesus macaque and dog see MGI.
Also for all species known gene tree, see Treefam database.
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, the phosphoglycerate mutases (PGAMs) and the acid phosphatase family diverged from a common ancestor (Jedrzejas, 2000; Okar et al., 2001). Alignments of the bisphosphatase domain with PGM and acid phosphatase can be obtained at EBI. On the other hand, PFK-2 domain is related to a superfamily of mononucleotide binding proteins including adenylate kinase (AK) of E. coli., p21 Ras, EF-tu, the mitochondrial ATPase- β-subunits and myosin ATPase, all of them contain the Walker A and B motifs and have a similar fold (Rider et al., 2004).

Orthologs (from BLAST Local Alignment Tool)
Results from BLAST Local Alignment Tool are shown in Figure 5. Only the annotated proteins are reported, the predicted proteins appearing in the local alignment were excluded.
Comparison of the PFKFB2 cDNA sequence with the bovine and rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) heart isoforms shows 87-90% nucleotide and 92-95% amino acid identity (Sakata and Uyeda, 1990; Darville et al., 1991).

Genomic variants
There are 647 SNP variants described in PFKFB2 (see GeneCards).
The most SNP are found in non coding regions: 418 are presented in introns, 3 in splice donor variant, 107 in 3 UTR and 25 variant within a half kb of the end of gene and others.
Furthermore, 61 SNP are presented with the coding regions. The most of them are missense (31 variants) and also synonymous variants (19 variants) and only one frameshift.

49 somatic mutations in the PFKFB2 gene detected in patient tumor samples are collected in the COSMIC database.
Coding silent substitutions: 20, which represent 40.8% of the mutations described among all patients. Two of them have been found in two patients: c.1008C>G (p.T336T) and c.1419G>A (p.S473S).
Nonsense substitutions: 1, located in c.1051C>T (p.R351*).
Missense substitutions: 23, which represent 46.9% of the mutations described among all patients.
Deletions frameshift: 1, located in c.1044delT (p.F348fs*66).
Insertion frameshift: 1, located in c.703_407insT (p.Q235fs*37).
Deletion inframe: 2, located in c.28_30delAAC (p.N12delN) and in in c.82_84delTGT (p.C28delC).
Unknown mutation: 2, one of them located in c.376-2A>T and the other in c.840+1G>A.
No synonymous substitutions or chromosomal fusions in PFKFB2 gene have been described in any tumor sample.