|Topic:||Regulation of Glycolytic Flux by PFK-2 .|
|Details:|| The synthesis of F2,6BP is catalyzed by the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase, or commonly just PFK-2). PFK-2 in mammalian organisms is a homodimer. The PFK-2 kinase domain is related to the catalytic domain of adenylate kinase. The F-2,6-BPase domain of the enzyme is structurally and functionally related to the histidine phosphatase family of enzymes. In the context of the active enzyme homodimer the PFK-2 domains function together in a head-to-head orientation, whereas the F-2,6-BPase domains can function as monomers. The PFK-2 reaction is catalyzed in the N-terminal half of the enzyme subunit, whereas the FBPase-2 reaction is catalyzed in the C-terminal half. There are four PFK-2 isozymes in mammals, each coded by a different gene that expresses several isoforms of each isozyme. The four different isozymes are expressed in the liver, heart, brain (or placenta) and testis and each differs by the sequences of their bifunctional catalytic cores and their N-terminal amino acid sequences.
Each of the different PFK-2 genes has been characterized. The PFKFB1 gene, located on the X chromosome (Xp11.21) and composed of 17 exons, encodes the liver isozyme. The PFKFB2 gene is found on chromosome 1q32.1 and is composed of 16 exons that encode the heart isozyme. The PFKFB3 gene is located on chromosome 10p15.1 and is composed of 19 exons that encode the brain/placenta isoform. The PFKFB4 gene is located on chromosome 3p21.31 and is composed of 19 exons that encode the testis isozyme. High level expression of the PFKFB4 gene is also seen in cancer cells and in response to hypoxia. The regulatory sequences present in these four genes have been identified that are responsible for their long-term control by hormones and tissue specific transcription factors. The PFKFB1 and PFKFB2 genes are the most highly characterized of the four.
The PFKFB1 gene is composed of 17 exons spanning 60 kbp and encodes three different mRNAs as a result of alternative splicing and alternative promoter useage. These mRNAs, and their promoters, are called L, M and F. The three mRNAs differ in their 5' ends but share 12 common exons (exons 2-13), six of which encode the PFK-2 catalytic domain and six of which encode the F-2,6-BPase catalytic domain. The L-type exon 1 is found the the L mRNA and the M-type exon 1 is found in the M mRNA. There are two F-type mRNAs that are derived by the splicing of two non-coding exons to part of the M-type exon 1. The L-type exon 1 sequences included in the L type enzyme contain a serine residue (Ser32) that is the target of PKA-mediated phosphorylation (see below). The L mRNA is expressed in liver and white adipose tissue, the M mRNA is expressed in skeletal muscle and white adipose tissue, and the F mRNA is expressed in fibroblasts, proliferating cells, and fetal tissues.
The PFKFB2 gene is composed of 20 exons spanning 22 kb that encode at least four mRNAs as a result of alternative promoter usage. Exons 3-14 are very similar to those of the PFKFB1 gene that code for the core catalytic domain. Exon 15 contains several phosphorylation sites. How the distinct 5' ends relate to the three mRNAs (H1, H2 and H4) that give rise to the 58 kDa isoform and the mRNA (H3) that encodes the 54 kDa isoform, is as yet unknown. Additionally, none of these mRNAs are strictly heart-specific in their pattern of expression.
The PFKFB3 gene is composed of 16 exons. Alternative splicing of exon 15 and possibly differential promoter usage yields two main isoforms that differ by a short C-terminal sequence. These two different PFKFB3 isoforms are referred to as the ubiquitous isoform (uPFK-2; also called the constitutive form) and the inducible isoform (iPFK-2). The inducible isoform is expressed at very low levels in adult tissues but its expression is induced in tumor cell lines and by pro-inflammatory stimuli. The uPFK-2 isoform has the highest kinase/bisphosphatase activity ratio. Of potential clinical significance is the fact that studies of iPFK-2 function indicate that the adipose tissue enzyme may play a role in the concept of healthy obesity. The vast majority of obese individuals will develop type 2 diabetes (T2D) and various cardiovascular diseases such as atherosclerosis. However, it has always been a scientific and clinical curiosity that a small percentage of overweight or obese individuals do not develop these same symptoms, the so-called healthy obese. In addition, it is known that certain thinner individuals may develop the types of health problems more typical of those associated with obesity. When iPFK-2 expression is knocked-out in mice there is a reduction in diet-induced obesity but the negative consequences include an exacerbation of adipose tissue inflammation and enhanced insulin resistance. This observation led researchers to speculate that iPFK-2 expression may link metabolic and inflammatory responses and, therefore, could underlie the healthy obesity concept. Results from the converse experiment does indeed strengthen the idea of iPFK-2 underlying healthy obesity. When iPFK-2 is overexpressed in adipose tissue there results an increase in fat deposition in adipose tissue which equates with obesity. However, these mice have suppressed inflammatory responses along with improved insulin sensitivity in both adipose tissue and the liver. The latter being equated with healthy obesity.
Rapid, short-term regulation of the kinase and phosphatase activities of PFK-2 are exerted by phosphorylation/dephosphorylation events. The liver isoform is phosphorylated at the N-terminus on Ser32, adjacent to the PFK-2 domain, by PKA. This PKA-mediated phosphorylation results in inhibition of the PFK-2 activity while at the same time leading to activation of the F-2,6-BPase activity. In contrast, the heart isoform is phosphorylated at the C-terminus by several protein kinases in different signaling pathways, resulting in enhancement of the PFK-2 activity. One of these heart kinases is AMPK and this activity allows the heart to respond rapidly to stress conditions that include ischemia. Insulin action in the heart also results in phosphorylation and activation of the PFK-2 activity of the enzyme. This insulin-mediated effect is, in part, the result of the activation of PDK1 (PIP3-dependent protein kinase). For more information on the signaling pathways initiated by the actions of insulin go to the Insulin Functions page.
Under conditions where PFK-2 is active, fructose flow through the PFK-1/F-1,6-BPase reactions takes place in the glycolytic direction, with a net production of F1,6BP. When the bifunctional enzyme is phosphorylated it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate. The metabolic result of the phosphorylation of the bifunctional enzyme is that allosteric stimulation of PFK-1 ceases, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these two enzymes is gluconeogenic, producing F6P and eventually glucose.
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