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 V_max 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 V_max with no change in K_m 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 K_m 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 V_max with no change in K_m 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 K_m for Fru-6-P and by increasing the V_max without affecting FBPase-2. Ser 466 phosphorylation is responsible for the increase in V_max whereas both phosphorylations are necessary to decrease the K_m 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.

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).