Departament de Ciencies Fisiologiques, Campus de Ciencies de la Salut, Universitat de Barcelona, Feixa Llarga s\\\/n, E-08907, LHospitalet de Llobregat, Barcelona, Spain
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).
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).
Laura Novellasdemunt ; Àurea Navarro-Sabaté ; Anna Manzano ; Ana Rodríguez-García ; Ramon Bartrons
PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3)
Atlas Genet Cytogenet Oncol Haematol. 2013-03-01
Online version: http://atlasgeneticsoncology.org/gene/45932/pfkfb3-(6-phosphofructo-2-kinase-fructose-2-6-biphosphatase-3)