561
Biochem. J. (2004) 381, 561–579 (Printed in Great Britain)
REVIEW ARTICLE
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head
with a bifunctional enzyme that controls glycolysis
Mark H. RIDER*1 , Luc BERTRAND†, Didier VERTOMMEN*, Paul A. MICHELS‡, Guy G. ROUSSEAU* and Louis HUE*
*Hormone and Metabolic Research Unit, Université Catholique de Louvain and Christian de Duve Institute of Cellular Pathology, 75, Avenue Hippocrate, B-1200 Brussels,
Belgium, †Division of Cardiology, Université Catholique de Louvain, 55, Avenue Hippocrate, B-1200 Brussels, Belgium, and ‡Research Unit for Tropical Diseases,
Université Catholique de Louvain and Christian de Duve Institute of Cellular Pathology, 75, Avenue Hippocrate, B-1200 Brussels, Belgium
Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that
controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure–
function relationships in PFK-2 (6-phosphofructo-2-kinase)/
FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation
of Fru-2,6-P2 . The FBPase-2 domain of the enzyme subunit bears
sequence, mechanistic and structural similarity to the histidine
phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo1-kinase), but this proved not to be correct. Molecular modelling
of the PFK-2 domain revealed that, instead, it has the same fold as
adenylate kinase. This was confirmed by X-ray crystallography. A
PFK-2/FBPase-2 sequence in the genome of one prokaryote, the
proteobacterium Desulfovibrio desulfuricans, could be the result
of horizontal gene transfer from a eukaryote distantly related to
all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with
possibly only one of the domains active), indicates that fusion of
genes initially coding for separate PFK-2 and FBPase-2 domains
might have occurred early in evolution. In the enzyme homodimer,
the PFK-2 domains come together in a head-to-head like fashion,
whereas the FBPase-2 domains can function as monomers. There
are four PFK-2/FBPase-2 isoenzymes in mammals, each coded
by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified
which account for their long-term control by hormones and tissuespecific transcription factors. One of these, HNF-6 (hepatocyte
nuclear factor-6), was discovered in this way. As to short-term
control, the liver isoenzyme is phosphorylated at the N-terminus,
adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein
kinase), leading to PFK-2 inactivation and FBPase-2 activation. In
contrast, the heart isoenzyme is phosphorylated at the C-terminus
by several protein kinases in different signalling pathways, resulting in PFK-2 activation.
INTRODUCTION
The synthesis of Fru-2,6-P2 from Fru-6-P (fructose 6-phosphate) and MgATP is catalysed by PFK-2 (6-phosphofructo-2kinase), whereas its hydrolysis to Fru-6-P and Pi is catalysed
by FBPase-2 (fructose-2,6-bisphosphatase) [1–7]. The PFK-2
and FBPase-2 reactions are catalysed on the same polypeptide of a
homodimeric protein. The PFK-2 reaction is catalysed in the
N-terminal half of the enzyme subunit, whereas the FBPase-2 reaction is catalysed in the C-terminal half [6,7]. PFK-2/FBPase-2
isoenzymes were first described in mammalian tissues and one
distinguishing feature is their PFK-2/FBPase-2 activity ratio. The
skeletal muscle isoenzyme has a V max of FBPase-2 5–10 times
greater than that of PFK-2, and in this respect resembles the
liver isoenzyme after phosphorylation by PKA (cAMP-dependent
protein kinase) [2]. When assayed with physiological concentrations of substrates, the heart [10] and testis [11] isoenzymes
possess a higher PFK-2/FBPase-2 activity ratio than the liver isoenzyme [2,4] and a molecular basis for this difference was recently
proposed [12] (see below).
In the present review, we have clarified the classification of
the mammalian PFK-2/FBPase-2 isoenzymes/isoforms, which is
now based on their gene origin rather than on tissue distribution.
Fru-2,6-P2 (fructose 2,6-bisphosphate) is found in all mammalian
tissues, throughout the animal and plant kingdoms, and in fungi
and certain unicellular eukaryotes, but not in bacteria (however,
see the section on evolution below) (for earlier reviews see [1–
7]). In most of these organisms, this molecule is a potent positive
allosteric effector of PFK-1 (6-phosphofructo-1-kinase) (except
in some protists in which it is an allosteric stimulator of pyruvate
kinase – see below) and thus stimulates glycolysis. In liver, Fru2,6-P2 is an inhibitor of FBPase-1 (fructose-1,6-bisphosphatase),
a regulatory enzyme of gluconeogenesis. Glucagon decreases
the concentration of hepatic Fru-2,6-P2 , thereby relieving the
inhibition of FBPase-1 and allowing gluconeogenesis to prevail.
Therefore Fru-2,6-P2 plays a unique role in the control of glucose
homoeostasis by allowing the liver to switch from glycolysis
to gluconeogenesis. In most mammalian tissues, which do not
contain FBPase-1, Fru-2,6-P2 acts as a glucose signal to stimulate
glycolysis when glucose is available. In heart, insulin and anoxia
increase Fru-2,6-P2 concentrations, which contributes to the
stimulation of glycolysis under these conditions [8,9].
Key words: catalysis, evolution, fructose 2,6-bisphosphate, gene,
glycolysis, phosphorylation.
Abbreviations used: ACC, acetyl-CoA carboxylase; AK, adenylate kinase; AMPK, AMP-activated protein kinase; Ca/CAMK, Ca2+ /calmodulin-activated
protein kinase; C/EBP, CCAAT/enhancer-binding protein; EGF, epidermal growth factor; FBPase-1, fructose-1,6-bisphosphatase; FBPase-2, fructose-2,6bisphosphatase; Fru-6-P, fructose 6-phosphate; Fru-2,6-P2 , fructose 2,6-bisphosphate; GAP, GTPase-activating protein; GLUT4, glucose transporter 4;
GR, glucocorticoid receptor; GRU, glucocorticoid-response unit; HNF, hepatocyte nuclear factor; MAPK, mitogen-activated protein kinase; NF-1, nuclear
factor 1; OC, Onecut; PDK, phosphoinositide-dependent protein kinase; PEPCK, phosphoenolpyruvate carboxykinase; PFK-1, 6-phosphofructo-1-kinase;
PFK-2, 6-phosphofructo-2-kinase; iPFK-2, inducible PFK-2; PI3K, phosphoinositide 3-kinase; PKA, cAMP-dependent protein kinase; PKB, protein kinase B;
PKC, protein kinase C; p70S6k , p70 ribosomal protein S6 kinase; 3D, three-dimensional; WISK, wortmannin-sensitive, insulin-stimulated protein kinase.
1
To whom correspondence should be addressed (e-mail [email protected]).
c 2004 Biochemical Society
562
Figure 1
M. H. Rider and others
Domain organization of the subunit sequences of some of the PFK-2/FBPase-2 isoenzymes
The N-terminal PFK-2 domain is shown in red and the C-terminal FBPase-2 domain is in blue. Numbering refers to the individual isoenzyme sequences. The black box in the PFK-2 domain indicates the
position of the glycine-rich loop in the nucleotide-binding fold with the signature G(X)4 GKT/S. The black box in the FBPase-2 domain contains the active site histidine in the RHG motif which has
become mutated in some of the monofunctional isoforms. Phosphorylation sites for protein kinases are indicated in red (see text).
PFK-2/FBPase-2 isoenzymes have also been identified in birds,
amphibians, worms, fish, insects, plants, fungi and trypanosomatids. Figure 1 shows the organization of domains in some of the
PFK-2/FBPase-2 isoenzymes, particularly those with unusual extensions or truncations and those which are controlled by phosphorylation. In the present review, we discuss the evolution of
the bifunctional enzyme, which seems to have arisen by gene
fusion. However, in trypanosomatids and yeasts, monofunctional
isoenzymes have evolved in which one or the other catalytic
domain has become inactive.
FBPase-2 is a classical Ping Pong reaction with formation of a
phosphoryl-enzyme intermediate on a histidine residue [6,7]. This
histidine residue is located in an RHG (Arg-His-Gly) motif, which
is characteristic of the so-called ‘histidine phosphatase’ family
(histidine is indicated in bold) of enzymes, including the acid
phosphatases and phosphoglycerate mutases. Indeed, the homology with the histidine phosphatases helped in our understanding
c 2004 Biochemical Society
of the structure and catalysis in the FBPase-2 domain, which has
been extensively reviewed elsewhere [6,7] and is not covered in
the present review. On the other hand, the PFK-2 mechanism has
proved more elusive and the structure–activity relationships in
the PFK-2 domain is one of the subjects of the present review.
Lastly, we consider how phosphorylation controls the activity
of the bifunctional enzyme. The heart isoenzyme is particularly
interesting as it is multisite-phosphorylated, integrating signalling
from many pathways via protein kinase cascades to a single molecule, Fru-2,6-P2 , to stimulate glycolysis.
THE PFK-2/FBPase-2 GENES IN MAMMALS
In mammals, and in keeping with the phylogenetic analysis (see
below), there are four PFK-2/FBPase-2 isoenzymes, namely the
liver, heart, brain (or placenta) and testis isoenzymes, which differ
by the sequence of their bifunctional catalytic core. They have
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
Table 1
The PFKFB2 gene
Mammalian FPK-2/FBPase-2 gene products
All the mRNAs listed, except M and F, differ in terms of coding sequence. The M isoform is
sometimes called the muscle isoenzyme. Only the mRNAs from gene 1 (formerly called gene A)
are referred to in the literature as indicated. We propose here an ad hoc nomenclature for the
other mRNAs. As gene 2 was called gene B, its transcripts were referred to earlier as B1 to B4.
Some of the isoforms are coded by more mRNAs than indicated, due to transcription from several
promoters and due to differential splicing with non-coding exons. See text for explanations and
references.
Gene
Chromosome locus
Isoenzyme
mRNA
Isoform
PFKFB1
Human Xp11.21
Rat Xq22-q31
Liver
PFKFB2
Human 1q31
Rat 13
Human 10p14-p15
Rat 17q12.3
Human 3p21-p22
Rat 8q32
Heart
L
M
F
H1, H2, H4
H3
U
I
T
L
M
M
Long (58 kDa)
Short (54 kDa)
Ubiquitous
Inducible
T
PFKFB3
PFKFB4
Brain/placenta
Testis
563
This gene codes for the heart isoenzyme and its two isoforms.
The rat gene (22 kb, 20 exons) [14,20] contains 12 consecutive
exons (3–14), which are very similar to those of the PFKFB1
gene that code for the core catalytic domain. Exon 15 contains
several phosphorylation sites for protein kinases. In bovine heart,
alternative splicing with deletion of exon 15 gives rise to an
additional, shorter (54 kDa instead of 58 kDa) isoform that lacks
the sequence coded by this regulatory exon [21,22]. The PFKFB2
gene gives rise, from distinct promoters, to at least four mRNAs
that differ by non-coding sequences at the 5 end [20,23]. How
these distinct 5 ends relate to the three mRNAs (H1, H2 and H4)
that give rise to the 58 kDa isoform and the mRNA (H3) for the
54 kDa isoform, is unknown. Moreover, none of these mRNAs
are strictly heart-specific [20]. For instance, the heart isoenzyme
is sometimes called the kidney isoenzyme.
The PFKFB3 gene
been named after the tissue from which they were first purified,
but they are expressed in other tissues as well, except for the testis
isoenzyme. A number of isoforms have been described for each
of these isoenzymes. The isoforms share the catalytic core of the
parent isoenzyme, but differ by flanking sequences, which often
contain sites for phosphorylation by protein kinases. As these additional sequences also influence the catalytic properties and
PFK-2/FBPases-2 ratio, some isoforms, e.g. liver and muscle,
have been considered as distinct isoenzymes.
A prerequisite for understanding the origin of the PFK-2/
FBPase-2 isoenzymes and isoforms, their tissue-specific expression, and their long-term control by hormones and growth factors was to clone the corresponding gene(s). Four genes have
been characterized in mammals, one for each isoenzyme (Table 1).
They are called PFKFB1 (also called gene A, located on human
chromosome X) [13], PFKFB2 (gene B, on human chromosome 1) [14], PFKFB3 (on human chromosome 10) [15] and
PFKFB4 (on human chromosome 3) [16]. These genes all have a
similar organization, which explains how each one codes for one
isoenzyme, for several isoforms by differential splicing, and for
even more mRNAs because of the presence of several promoters
and 5 non-coding exons.
The PFKFB1 gene
This gene (60 kb, 17 exons), which codes for the liver isoenzyme,
gives rise, from distinct promoters (called L, M and F from 3
to 5 ), to the L, M and F mRNAs respectively [13,17,18]. These
three mRNAs differ by their 5 end and share 12 consecutive exons
(exons 2–13) corresponding to the catalytic core, six coding for
the PFK-2 domain, followed by six for the FBPase-2 domain. An
additional 5 coding exon (exon1L ) is present in the L mRNA,
which codes for the L isoform, and another one (exon1M ) in the
M mRNA, which codes for the M isoform. The two F mRNAs,
which contain two non-coding exons upstream from, and spliced
with part of, exon1M also code for the M isoform. Thus the M
isoform has the same sequence as the L isoform except for the
N-terminus, where the first 32 residues, including the PKA-phosphorylated Ser32 , are replaced by an unrelated nonapeptide devoid
of phosphorylation sites. The L isoform, expressed from the L promoter, is the main PFK-2/FBPase-2 in liver; it is also found in
white adipose tissue [19]. The M isoform is expressed from the
M promoter in skeletal muscle and white adipose tissue, and from
the F promoter in fibroblasts, foetal tissues and proliferating cells.
This gene, which contains at least 16 exons, codes for an isoenzyme originally cloned from bovine brain [24] and from human
placenta [25]. Alternative splicing of exon 15 and possibly differential promoter usage yields two main isoforms that differ by a
short C-terminal sequence [15]. These are the so-called ubiquitous
(or constitutive) isoform [26] and the inducible isoform [27],
the expression of the latter being very low in adult tissues, high
in tumour cell lines and increased by pro-inflammatory stimuli.
Additional splice variants of this gene have been described: six in
rat brain [28] and four in human brain [29].
The PFKFB4 gene
The testis isoenzyme [16,30] is encoded by the PFKFB4 gene,
which has been characterized in humans [16]. The expression of
this isoenzyme, possibly translated from two mRNAs that share
the same coding sequence, is reportedly testis-specific [16].
THE PFK-2 REACTION: CATALYSIS, SITE-DIRECTED
MUTAGENESIS AND MODELLING
Kinetic studies of product inhibition [31,32] and stereochemical
investigations [33] showed that the PFK-2 reaction involves ternary complex formation with direct in-line transfer and inversion
of configuration of the γ -phosphate of ATP. Based on a sequence
alignment, Pilkis and co-workers speculated that the PFK-2
domain was analogous to bacterial PFK-1 [34,35]. Accordingly,
Cys160 of the PFK-2 domain (numbering refers to the rat liver
isoenzyme unless specified otherwise), corresponding to Asp127 in
bacterial PFK-1, would act as a base catalyst. This would explain
why the kcat of PFK-2 is approx. 1000-times less than that of
PFK-1, since a cysteine residue is expected to be a much poorer
base catalyst than an aspartate residue. However, our site-directed
mutagenesis experiments indicated that mutation of Cys160 to
aspartic acid or serine barely affected the kcat [36], arguing against
the PFK-1 analogy. A closer inspection of the PFK-2 sequence
revealed two regions containing the so-called ‘Walker A’ and
‘Walker B’ motifs or nucleotide-binding fold [37,38]. These motifs are found in the AK (adenylate kinase), Ras and EF-Tu family
of nucleotide binding proteins [39,40]. Indeed, mutagenesis of
Lys54 and Thr55 [37] in the Walker A motif, and Asp130 [38] in the
Walker B motif drastically decreased PFK-2 activity. Multiple sequence alignments of the PFK-2 domains of the PFK-2/FBPase-2
isoenzymes using the ‘Matchbox’ algorithm revealed conserved
boxes of residues and mutagenesis of residues within these boxes
c 2004 Biochemical Society
564
Figure 2
M. H. Rider and others
For legend, see facing page
c 2004 Biochemical Society
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
allowed us to pinpoint residues that are important for substrate
binding and catalysis [41–44]. An important residue for catalysis
was identified as Lys174 [43]. In the yeast fbp26 isoenzyme, which
lacks PFK-2 activity, this amino acid is replaced by a glycine
residue. Indeed, mutation of Lys174 to glycine in the PFK-2 domain
of the liver isoenzyme drastically reduced the kcat [43], and it
is likely that this residue, together with Thr55 of the Walker A
motif, stabilizes the transition state of the PFK-2 reaction during
phosphate transfer (see below). A summary of the residues that
are important for the PFK-2 reaction that we, and others, have
identified as being important for substrate binding and catalysis,
is shown in Figure 2(A).
Sequence similarities were found between the PFK-2 domain
and other nucleotide-binding proteins, the most similar being AK
[41]. Sequence alignments and secondary-structure prediction allowed us to propose a structure model of the PFK-2 domain, based
on the 3D (three-dimensional) structure of AK [41]. The model
was consistent with the roles of amino acid residues proposed from
site-directed mutagenesis studies and was confirmed in the 3D
structure of the testis isoenzyme, solved by Uyeda and co-workers
[45]. The crystal structure of this isoenzyme contained an ATP
analogue, but the other substrate, Fru-6-P was not present. Initial
mutagenesis experiments suggested that Arg195 [46] and Arg104
[47] are important for Fru-6-P binding. Using the X-ray coordinates of the testis isoenzyme, we were able to identify
residues in the Fru-6-P-binding pocket by computer docking
(Figure 2C) and the role of these residues was confirmed by
site-directed mutagenesis [44]. These studies indicate that five
arginine residues are involved in Fru-6-P binding (Figure 2A).
Although the PFK-2 domain displays structural similarity with
AK, the kcat of PFK-2 (at best, approx. 0.1 s−1 ) is more than three
orders of magnitude less than that of AK [48]. Another family
member, the small G-protein Ras, has evolved to maintain a low
catalytic activity [49] (kcat 1000 times lower than that of PFK-2),
due to a highly flexible and mobile active site. However, in the
presence of the GTPase-activating protein GAP, the kcat is increased 105 -fold to about 20 s−1 [50]. The catalytic mechanism of
Ras was originally proposed to involve proton abstraction from
water by a general base, Gln61 . This would help to explain the low
kcat of Ras, since a glutamine residue would be expected to be a
poor base catalyst. Indeed, replacement of Gln61 by glutamic acid
increased the rate of GTP hydrolysis 20-fold [49]. However, an
alternative mechanism for Ras-catalysed GTP hydrolysis has been
proposed (substrate-assisted catalysis) [51]. In this mechanism,
it is the γ -phosphoryl group of GTP, the substrate, that acts as a
base catalyst. A central question in the understanding of phosphotransferase reaction mechanisms is whether they operate principally via an associative (SN 2-like) or dissociative (SN 1-like) transition state [52–54]. For the PFK-2 reaction, the two mechanisms
are shown in Figure 3. If a negative charge develops on the γ -phosphate oxygens, the reaction is associative and occurs via a pentacoordinated phosphoryl intermediate. In a dissociative mechanism, a
negative charge arises on the leaving group, which for the PFK-2
reaction is the β–γ -bridging oxygen atom of ADP. The Rascatalysed hydrolysis of GTP was suggested to be associative,
with Gln61 stabilizing the transition state by correctly positioning
the attacking water molecule [55]. However, others have made
565
Figure 3 Associative and dissociative transition states for phosphotransferase catalysis in PFK-2
In the single displacement reaction, both substrates must be bound to the enzyme forming a
ternary complex. Phosphoryl transfer can then take place either via (A) an associative transition
state (SN 2-like) or (B) a dissociative transition state (SN 1-like).
arguments in favour of a dissociative transition state [54]. GAP
accelerates the GTPase reaction by introducing the so-called
‘arginine finger’ (Arg789 ) to stabilize further the β–γ -bridge oxygen [56]. For the PFK-2 domain, all attempts to identify a residue that could fulfil the role of a base catalyst by site-directed
mutagenesis have failed. It is noteworthy that catalysis in AK
and related kinases does not seem to involve a base catalyst. The
position of Lys174 in the PFK-2 domain structure [45] is such that
it could stabilize a negative charge on the β–γ -bridge oxygen of
ATP, favouring a dissociative transition state, in a manner similar
to the arginine finger of GAP in Ras-catalysed GTP hydrolysis
[54]. The low kcat of PFK-2 could be due to either a ‘loose’ transition state or the two substrates being far away from each other.
STRUCTURE OF PFK-2/FBPase-2
The crystal structure of the rat testis isoenzyme [45] confirmed
that the PFK-2 domain is related structurally to the AK/Ras family,
whereas the FBPase-2 domain is related to the histidine phosphatases. In the homodimeric structure [45], the two PFK-2 domains
come together in a head-to-head fashion, whereas the FBPase-2
domains are basically independent (Figure 4A), with only one
connecting salt bridge. The structure contained two bound phosphates, indicating the position of the FBPase-2 active site, and a
molecule of ATPγ S in the PFK-2 active site ([45], and see Figure 5). The PFK-2 domain is a six-stranded β-sheet surrounded
Figure 2 Residues involved in catalysis and substrate binding in the PFK-2 domain (A), stereo view of the location of critical residues for ATP-binding and
catalysis (B) and Fru-6-P binding (C) in the 3D structure of the testis isoenzyme
The numbering of residues refers to the liver isoenzyme sequence. In (A), residues involved in substrate binding and catalysis in the PFK-2 domain are shown. In (B) and (C), the X-ray co-ordinates
were retrieved from the PDB database (accession code 1BIF) corresponding to the testis isoenzyme containing ATPγ S in the PFK-2 domain. The position of Fru-6-P was modelled as described [44].
The trace of the backbone is shown in green. Substrates and side-chains of critical residues are represented with carbon atoms in white, oxygen in red, nitrogen in blue and phosphate in yellow.
Distances indicated by dotted lines are in Å.
c 2004 Biochemical Society
566
M. H. Rider and others
Figure 5 3D structure of one enzyme subunit of the testis PFK-2/FBPase-2
isoenzyme
The co-ordinates were retrieved from the PDB database (accession code 1BIF) containing ATPγ S
in the PFK-2 domain. The position of Fru-6-P was modelled as described in [44]. In the upper
right hand PFK-2 domain, ATP is on the left and Fru-6-P is on the right. In the lower left hand
FBPase-2 domain, two inorganic phosphates indicate the position of the Fru-2,6-P2 -binding
site. The PFK-2 domain is composed of a β-sheet surrounded by α-helices. Two subdomains,
composed of α-helices (above) form a flexible cover and are involved in Fru-6-P binding and
catalysis (induced fit, see text).
Figure 4 3D structure of the testis PFK-2/FBPase-2 homodimer (A) and
close-up view of the head-to-head dimer interface (B)
(A) The co-ordinates were retrieved from the PDB database (accession code 1BIF). In this Figure,
the PFK-2 domains are in green and come together in a head-to-head like fashion. The FBPase-2
domains are below in white. The α7 helices in each subunit are indicated by green arrows, and
the α13 helices are in red (see text). The C-terminal ends in each subunit are indicated by yellow
arrows. The side chains of residues in the PFK-2 domain shown in (B) are also shown in (A)
and are coloured white in the upper domain. (B) Top view of the head-to-head interaction seen
after turning the structure through 90 ◦ towards the reader along the plane of the β-sheet. Part of
the interacting continuous 12-stranded β-sheet is shown together with the positions of some
of the residues that differ between the testis and liver isoenzymes, namely Ser225 (replaced by
arginine in the liver isoenzyme), Gln224 (replaced by threonine in the liver isoenzyme), along
with Glu208 , which is conserved (see text).
by seven α-helices (Figure 5). The Walker A motif is located
at the C-terminal end of the first β-strand and the phosphates
of ATPγ S are bound in the anion hole of the phosphate-binding
loop. Lys54 of the Walker A motif interacts with both the β- and
γ -phosphates of ATP (Figure 2B), while Thr55 and Asp130 (Walker
B motif) provide two ligands for the octahedral Mg2+ ion, another
two being provided by the β- and γ -phosphates of ATP [45]. ATP
is also bound by the stacking of the adenine ring against non-polar
side chains. Although the crystal structure was devoid of Fru-6-P,
its binding site was predicted to be analogous to the AMP site in
c 2004 Biochemical Society
AK [44], with the 6-phosphate group of Fru-6-P bound by Arg104 ,
Arg138 and Arg195 (Figure 2C).
The two central six-stranded β-sheets of the PFK-2 domains
interact to form a continuous twelve-stranded inter-monomer βsheet [45], part of which is shown in Figure 4(B). Therefore
PFK-2 functions as a homodimer, in contrast with AK, which,
in mammals, is a 22 kDa monomer. Substrate-induced conformational changes have been proposed for kinases such as hexokinase
[57] and AK [58]. AK contains a cleft at the active site. ATP and
AMP bind at opposite ends of this cleft with their phosphates
pointing towards each other and lying in the centre of the cleft.
Substrate binding causes a switch to the structure in which the
cleft closes [58]. By analogy with AK, the PFK-2 domain is predicted to contain two mobile segments which trap Fru-6-P and
ATP [45], thereby excluding water to prevent ATP hydrolysis.
Therefore the PFK-2 domain is a bi-lobed structure (Figure 5)
and induced-fit changes on substrate binding are a likely feature,
in common with other kinases.
Recently, the crystal structure of the human liver PFK-2/
FBPase-2 isoenzyme was solved [12]. Superimposition of the
structures of the liver and testis isoenzymes indicated that the overall folding was similar, as expected. However, the sequence differences (26 %) cause conformational differences, including a
twist of the FBPase-2 domain relative to the PFK-2 domain. This
allows new dimeric interactions between the FBPase-2 domains of
the liver isoenzyme, increasing their conformational stability and
FBPase-2 activity. Sequence differences in the FBPase-2 binding
pocket of the liver isoenzyme would also explain its higher affinity
for Fru-2,6-P2 relative to the testis isoenzyme. Like in the testis
isoenzyme, the PFK-2/FBPase-2 interface of the liver isoenzyme
includes hydrophobic interactions between a β-hairpin (residues
215–230) and the following helix α7 of the PFK-2 domain, which
in turn interacts with helix α13 in the FBPase-2 domain (Figure 4A). Part of this structure also forms the dimeric interface
by which the PFK-2 domains come together head-to-head
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
(Figure 4B). Sequence differences in this hairpin (T224Q, A225S
and Q232A, liver compared with testis isoenzyme; numbering
refers to the human liver isoenzyme) could impart a twist
in the FBPase-2 domain [12]. Arg225 in the liver isoenzyme
forms a salt bridge with Glu208 (see Figure 4B) participating in the
rotation of the hairpin structure relative to the testis isoenzyme,
a rotation that triggers the swing of the FBPase-2 domains.
Interestingly, we showed that Arg225 in the liver isoenzyme was
labelled by the group-specific reagent, phenylglyoxal, and proposed that this residue might be involved in Fru-6-P-binding
in the PFK-2 domain [59]. Indeed, mutation of Arg225 to serine in
the liver isoenzyme increased the K m of PFK-2 for Fru-6-P
and decreased the V max [47]. However, the Arg225 → Ser mutant
displayed an increase in the V max of FBPase-2, contrary to its proposed role from the crystal structure. This might be because
other hydrogen-bonding interactions via Thr224 and Gln232 would
be maintained and that mutation of all three residues would be
necessary to change the kinetics of FBPase-2 into those characteristic of the testis isoenzyme. Lastly, it is perhaps noteworthy
that phosphorylation by PKA decreased the labelling and PFK-2
inactivation by phenylglyoxal [59]. An interesting possibility is
that the phosphorylated Ser32 might interact with Arg225 and in
some way transmit conformational changes to the FBPase-2 and
PFK-2 domains (see below).
EVOLUTION OF PFK-2/FBPase-2
Mammalian PFK-2/FBPase-2 sequences have been derived from
cDNAs obtained from liver (rat [60,61], human [62,63], bovine
[64] and mouse [65]), skeletal muscle (rat [66]), heart (rat [67],
bovine [68], human [23] and mouse [65]), placenta/brain (human
[25,69], bovine [24] and rat [70]) and testis (human [16] and rat
[11]). Sequences are also available from birds (chicken liver [71]),
amphibians (frog muscle and frog liver [72]), fish (seabream,
Sparus aurata [73]), plants (Arabidopsis thaliana [74], potato
[75] and spinach [76]), baker’s yeast [77–79] and from various
genome projects, such as Caenorhabditis elegans, Drosophila
melanogaster, Neurospora crassa, Schizosaccharomyces pombe
and trypanosomatids. Interestingly, a search of 272 eubacterial
genomes in the NCBI database with the full-length rat liver
PFK-2/FBPase-2 isoenzyme sequence revealed the presence of
a homologue in Desulfovibrio desulfuricans (see below). The
FBPase-2 domain, the phosphoglycerate mutases and the acid
phosphatases probably diverged from a common ancestor
[6,7,34,35], whereas the PFK-2 domain is related to a superfamily
of mononucleotide binding proteins including AK, p21 Ras,
EF-Tu, the mitochondrial ATPase β-subunits and myosin ATPase,
all of which contain the Walker A and B motifs and have a similar fold ([41,45] and see above). As most of the PFK-2/FBPase-2
sequences contain an N-terminal PFK-2 domain and a C-terminal FBPase-2 domain in the same polypeptide, the bifunctional
enzyme must have arisen by gene fusion followed by duplication
of this primordial gene. The PFK-2 and FBPase-2 domains can
be flanked by variable extensions, which, in the mammalian
isoenzymes, correspond to regulatory domains often containing
phosphorylation sites for protein kinases. The plant [74–76],
Neurospora and yeast pfk26 [77] sequences possess a long
N-terminal end responsible for an increase in molecular mass of
the enzyme subunits from about 55 kDa to 90 kDa. The two catalytic cores have also evolved into inactive domains in lower organisms. For example, the yeast pfk26 is devoid of FBPase-2
activity, principally by mutation of the active site histidine in the
RHG motif to serine [77], while yeast fbp26 lacks PFK-2 activity
([79], and see above). The yeast pfk27 is essentially a PFK-2 do-
567
main (subunit molecular mass of approx. 40 kDa) [78] with a
small C-tail (Figure 1).
Fru-2,6-P2 and PFK-2 and FBPase-2 activities have also been
detected in other simple eukaryotes: in the unicellular photosynthetic flagellate Dunaliella sp. (phylum Chlorophyta) and in
the slime mould Dictyostelium discoideum (only Fru-2,6-P2 detected) (reviewed in [5]). No Fru-2,6-P2 , PFK-2 or FBPase-2 could
be detected in the protists Tritrichomonas foetus and Trichomonas
vaginalis (phylum Parabasalia) and Isotricha prostoma (phylum
Ciliaphora) [5]. In trypanosomatids, most of the glycolytic pathway is compartmentalized within the glycosome, where PFK-1
is insensitive to Fru-2,6-P2 [80]. In contrast, cytosolic pyruvate
kinase is stimulated by Fru-2,6-P2 , consistent with the presence of
PFK-2 and FBPase-2 in the cytosol [80]. By searching trypanosomatid databases, four genes potentially encoding PFK-2/
FBPase-2 isoenzymes were found in both Trypanosoma brucei
and Leishmania major, and were termed Tb1/Lm1 to Tb4/Lm4
(N. Chevalier, L. Bertrand, M. H. Rider, F. R. Opperdoes, D. J.
Rigden and P. A. M. Michels, unpublished work). Lm1, Tb1 and
Lm4 are predicted to contain long N-terminal extensions, whereas
the molecular masses of Lm2, Lm3 and Tb3 are predicted to be
more akin to the subunit molecular masses of the mammalian isoforms. Tb1 and Lm1 would possess PFK-2 activity and expressed
recombinant Tb1 was indeed active. The PFK-2 domain of Lm2
could be active, although some important residues for catalysis (Lys54 , Asp130 and Arg195 ) are mutated. The FBPase-2 domains of Tb1, Lm1 and Lm2 contain several mutated key residues
and would be inactive. Therefore Tb1, Lm1 and Lm2 would be
monofunctional PFK-2 isoenzymes. Lm3 and Tb3, on the other
hand, are predicted to be monofunctional FBPase-2s. Lm4 and
Tb4 would be inactive in the PFK-2 domains, but could have
FBPase-2 activity, where the role of the nucleophilic histidine
residue in the RHG motif, replaced by proline or asparagine respectively, would be fulfilled by another residue.
To look more closely at evolutionary relationships, phylogenetic trees were made from alignments of the separate PFK-2 and
FBPase-2 catalytic core sequences taken from databases with
AK and phosphoglycerate mutase as outgroups respectively (Figure 6). The mammalian isoenzymes cluster into the liver, heart,
brain/placenta and testis groups, reflecting the four different
genes, while in plants, only a single gene has been detected. The
evolution of PFK-2/FBPase-2 in yeasts and trypanosomatids is
not easy to deduce from the phylogenetic trees. This is due to low
levels of conservation in the two domains and the fact that in these
organisms, one or both domains have been inactivated and possibly subjected to a high evolution rate. Most of the isoenzymes
of fungi have probably arisen from gene duplications within that
group. Also, in the tree of the FBPase-2 domain, all the trypanosomatid sequences cluster together, separated from the sequences
of all other organisms (but containing the outgroup). The cluster
comprises two sets of two subgroups containing putative PFK-2s
(Lm1/Tb1/Tc1 and Lm2; Tc1 is from Trypanosoma cruzi) and
FBPase-2s (Lm3/Tb3 and Lm4/Tb4) respectively. The situation
for trypanosomatids in the PFK-2 tree is very similar, except for
the presence of yeast pfk27 in the cluster. The phylogenetic analysis indicates that the four isoenzymes in trypanosomatids
already existed in the ancestor of Trypanosoma and Leishmania.
A PFK-2/FBPase-2 sequence in the completed genome of
Desulfovibrio desulfuricans raises interesting possibilities as to
the evolutionary origin of the bifunctional enzyme, assuming that
its presence is not due to contamination. The gene neighbours of
the putative bifunctional enzyme in Desulfovibrio are genuinely
bacterial with no evidence that a eukaryotic DNA could have
been introduced. The sequence would be expected to code for
an enzyme that would be functional in both domains, based on
c 2004 Biochemical Society
568
Figure 6
M. H. Rider and others
Phylogenetic trees constructed from sequences of the separate PFK-2 and FBPase-2 domains
Sequences of PFK-2 and/or FBPase-2s were aligned using the computer program ClustalX [208]. From this alignment, sub-alignments of either the PFK-2 domain or the FBPase-2 domain were
made. The residues were from 42 to 248 for the PFK-2 domain and from 249 to 438 for the FBPase-2 domain (numbering refers to the rat liver isoenzyme). The sequences were yeast PFK-2 27
c 2004 Biochemical Society
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
the conservation of residues for substrate binding and catalysis.
Desulfovibrio species are strict anaerobes that can oxidize
low-molecular-mass compounds such as lactate, pyruvate, fumarate, ethanol and glycerol. However, they can also store and synthesize polyglucose which can be catabolized in the absence of
exogenous substrates, and the resulting NADH can be utilized
for the reduction of sulphate, thiosulphate or nitrite. It remains to
be seen whether Fru-2,6-P2 would be present in these organisms,
for example to control PFK-1 during polyglucose degradation.
The gene does not seem to be part of an operon, although the downstream neighbour is 4-α-glucanotransferase (amylomaltase). The
fact that a PFK-2/FBPase-2 is found in only a single bacterium
out of over 300, also including archaea, makes it unlikely that the
bifunctional enzyme arose in a bacterium. Its existence could be
due to horizontal gene transfer from a eukaryote. The phylogenetic
trees show that the Desulfovibrio sequences branch at the root of
the tree and are not close to any of the other organisms (Figure 6).
Therefore the donor of the gene was most likely to be a protist
unrelated to trypanosomatids. The presence of a PFK-2/FBPase-2
sequence in Desulfovibrio, with its likely origin from a protist,
and the predicted domain structure of the isoenzymes in the trypanosomatids, indicates that the gene fusion giving rise to a bifunctional enzyme occurred early in eukaryotic evolution in an
ancestral unicellular organism.
The occurrence of multiple isoforms of PFK-2/FBPase-2 in
mammals can be understood as a need for enzymes with different kinetic and regulatory mechanisms to regulate glycolysis
(gluconeogenesis) in different tissues under various hormonal and
nutritional states. In yeast, pfk26 is activated by PKA [77,82],
whereas the synthesis of pfk27 is induced by fermentable carbon
sources [78]. Neither is essential for growth [78,83], but there is
evidence for distinct roles for the two yeast PFK-2s from metabolomic analysis of mutants [84]. The reason for the existence of
four isoenzymes in trypanosomatids could be to adapt to radically
different nutritional environments encountered by the parasite in
its complicated life cycle between insects and mammals. Why
the bifunctional PFK-2/FBPase-2 evolved into monofunctional
enzymes in yeasts and trypanosomatids is unclear. It might represent an adaptation to the specific requirements of glucose metabolism in these unicellular organisms, different from the requirements in the ancestral eukaryote where the fusion of the PFK-2
and FBPase-2 domains in a single enzyme occurred. One may
wonder why, in the monofunctional enzymes, an inactive PFK-2
or FBPase-2 domain has been retained. Only in the yeast pfk27
has the FBPase-2 domain diverged considerably and become truncated. For the mammalian PFK-2/FBPase-2s, the PFK-2 domains
are intimately associated in the 3D structure (see above), whereas
the FBPase-2 domains are independent in the testis isoenzyme
[45], but they do form contacts in the liver isoenzyme [12]. Also,
when expressed separately, the PFK-2 domain forms inactive
aggregates [85], while the FBPase-2 domain can be active as
a monomer [86]. Therefore the inactive domains have been kept
probably to maintain the active dimeric structure of the enzyme.
569
For the mammalian isoenzymes, the advantages of bifunctionality
are simplicity in short-term control, such as by phosphorylation
or by allosteric effectors, and simplicity in long-term control (one
mRNA per bifunctional polypeptide). One reason why certain
micro-organisms at a later stage of evolution again uncoupled
PFK-2 and FBPase-2 could be to give increased flexibility to adapt
to different growth conditions. Different combinations of monofunctional PFK-2 and FBPase-2 enzymes might become expressed in trypanosomatids growing in different environments,
and this could be studied by following the expression of the
different enzymes throughout the life cycle of the parasite.
TRANSCRIPTIONAL CONTROL OF MAMMALIAN PFK-2/FBPase-2
Regulatory sequences that account for some of the mechanisms
involved in the long-term hormonal control and tissue-specific
expression of PFK-2/FBPase-2 have been identified in the
PFKFB1, PFKFB2 and PFKFB3 genes.
PFKFB1 gene
This gene has been extensively studied, since its expression is
stimulated by glucocorticoids, an effect inhibited by insulin, by
glucose and by mitogenic stimuli, and because its L promoter
is liver- and adipose-tissue-specific. Glucocorticoids and insulin
control PFKFB1 via a GRU (glucocorticoid-response unit) located in its first intron [87]. A combination of in vitro DNAbinding assays, transfection experiments and in vivo genomic
footprinting showed that this GRU behaves as an enhancer in
an open chromatin conformation [88] and binds the transcription
factor NF-1 (nuclear factor 1) in unstimulated cells. Binding of
glucocorticoids to their GR (glucocorticoid receptor) on the GRU
induces binding of HNF (hepatocyte nuclear factor)-3, which
then co-operates with the GR [89]. Insulin targets the ligandbinding domain of the GR, whose action is thereby inhibited, via a
signalling cascade that is distinct from the PI3K (phosphoinositide
3-kinase) or MAPK (mitogen-activated protein kinase) pathways
[90], but appears to involve the JNK (c-Jun N-terminal kinase)/
SAPK (stress-activated protein kinase) pathway [91].
The F promoter binds the ets (proto)oncogene product, which
stimulates its activity [17,18]. F promoter activity is inhibited
when cells differentiate, through its binding of the E2F transcription factor. F promoter activity is stimulated at the G1 /S
transition of the cell cycle by growth factors [e.g. EGF (epidermal
growth factor)] and oncogene (e.g. v-src and E1A) products, which
relieve the E2F-mediated inhibition [92]. This effect of EGF is
inhibited by cAMP and could involve MAPK [93] and PI3K/PKB
(protein kinase B) [94] signalling. Moreover, F precursor mRNA
splicing is blocked in G0 and this block requires the E2F-binding
site [95]. These data shed light on the mechanisms that switchoff transcription from the F promoter, e.g. in adipoblasts when
they differentiate into adipocytes, which use instead the M and
(no. Q12471), yeast PFK-2 26 (no. P40433), Neurospora crassa PFK-2/FBPase-2 (no. Q9P522), Schizosaccharomyces pombe PFK-2/FBPase-2 (nos. Q9UTE1, Q8TFH0), spinach (Spinacea
oleracea ) PFK-2/FBPase-2 (GenBank® accession no. AF041848), Arabidopsis thaliana PFK-2/FBPase-2 (GenBank® accession no. AF190739), potato (Solanum tuberosum ) PFK-2/FBPase-2
(GenBank® accession no. AF073830), Leishmania major PFK-2 designated ‘Lm1’ (no. Q9NKN9), yeast FBPase-2 26 (no. P32604), Caenorhabditis elegans PFK-2/FBPase-2 (no. Q21122), Drosophila
melanogaster PFK-2/FBPase-2 (no. Q9VWH7), human testis PFK-2/FBPase-2 (no. Q16877), rat testis PFK-2/FBPase-2 (no. P25114), bovine brain PFK-2/FBPase-2 (no. Q28901), human placenta
PFK-2/FBPase-2 (no. Q16875), mouse placenta PFK-2/FBPase-2 (no. Q9ESY2), rat brain PFK-2/FBPase-2 (no. O35552), bovine heart PFK-2/FBPase-2 (no. P26285), human heart PFK-2/FBPase-2
(no. O60825), mouse heart PFK-2/FBPase-2 (no. P70265), rat heart PFK-2/FBPase-2 (no. Q9JJH5), chicken liver PFK-2/FBPase-2 (no. Q91348), seabream liver PFK-2/FBPase-2 (GenBank®
accession no. U84724), bullfrog liver PFK-2/FBPase-2 (no. Q91309), rat liver PFK-2/FBPase-2 (no. P07953), bovine liver PFK-2/FBPase-2 (no. P49872), human liver PFK-2/FBPase-2 (no. P16118).
The trypanosomatid sequences were retrieved from the relevant genome databases. The Desulphovibrio desulfuricans sequence (accession no. ZP 00131027) was taken from a BLAST search of the
NCBI eubacterial genomes with the full-length rat liver PFK-2/FBPase-2 sequence. Accession numbers are for Swiss-Prot/TrEMBL unless stated otherwise. Phylogenetic trees were created using
the tree option of the ClustalX program after exclusion of positions with gaps. Arabidopsis thaliana AK (no. Q9ZUU1) and E. coli phosphoglycerate mutase (no. P36942) were taken as outgroups to
root the PFK-2 and FBPase-2 trees respectively. Horizontal bars represent ten substitutions per 100 residues.
c 2004 Biochemical Society
570
M. H. Rider and others
L promoters, and in myoblasts when they differentiate into adult
muscle, which uses the M promoter.
Glucose increases the concentration of the F and L mRNAs in
hepatic cell lines. This effect of glucose is mimicked by xylitol,
a precursor of intermediates of the pentose phosphate and glycolytic pathways, and is inhibited by okadaic acid, an inhibitor of
protein phosphatases. Transfection experiments showed that the
F promoter region is a target of the glucose effect. Another region
of the gene, located between the F and L promoters, behaves as a
glucose-sensitive enhancer of both the F and the L promoters. This
region corresponds to a cluster of DNase-I hypersensitive sites
that are induced in chromatin following glucose treatment [96].
Its sequence organization resembles that of the glucose-sensitive
insulin gene promoter. The transcription factor(s) involved in this
glucose effect have not been identified.
The regulation of the M promoter is poorly understood. Its
TATA box region binds the transcription factor C/EBP (CCAAT/
enhancer-binding protein), which stimulates its activity [97]. This
could play a role in white adipose tissue, since C/EBP is an
important transcriptional activator of genes that are expressed
in adipocytes. The DNA-binding and transactivation properties
of C/EBP are stimulated through its direct interaction with the
tumour suppressor protein Rb and this is crucial for C/EBPdependent terminal differentiation of fibroblasts into adipocytes.
Fibroblasts express PFK-2 mRNA from the F promoter and this
depends on the transcription factor E2F. The latter is inhibited
by Rb, which switches off the F promoter. Thus C/EBP could be
an important component of the F-to-M promoter switch during
differentiation [98].
The basal activity of the L promoter, which is controlled by
the GRU discussed above, was found to depend on its binding of
both ubiquitous (NF-1 and Oct-1) and liver-specific (HNF-3 and
C/EBP) transcription factors [99,100]. Further study of regulatory
protein binding to this promoter led to the discovery [100] and
cloning [101] of HNF-6.
HNF-6 is the prototype of a new class, called Onecut (OC)
[102], of homoeoproteins conserved from nematodes, ascidians,
flies and zebrafish to mammals, with three members in humans,
namely HNF-6 or OC-1, OC-2 and OC-3 [103,104]. In adult
liver, HNF-6 stimulates the transcription of genes coding for
secreted proteins [105], for P450 cytochromes [106] and for enzymes of glucose metabolism, including PFK-2/FBPase-2, PEPCK
(phosphoenolpyruvate carboxykinase) [107], glucose-6-phosphatase [108] and glucokinase [109]. Interestingly, the Hnf6 gene
is controlled by growth hormone [106,110]. HNF-6 can also
counteract the stimulatory action of glucocorticoids on transcription of the PFKFB1 and PEPCK genes, by interacting with
the GR on the DNA of these target genes [107]. As the enzymes
controlled by HNF-6 are involved in both gluconeogenesis and
glycolysis, the effect of HNF-6 might be to provide the hepatocyte
with an adequate enzyme level for short-term regulation by other
signals rather than influence the direction of carbohydrate metabolism. In any case, Hnf6-knockout mice are diabetic [109].
In the embryo, HNF-6 is expressed in developing liver and
pancreas, where it regulates genes coding for transcription factors
that control cell differentiation and morphogenetic programmes.
Studies on Hnf6-knockout mice showed that HNF-6 controls the
timing of pancreas specification from the endoderm by inducing
the transcription factor Pdx-1 [111]. Later on, HNF-6 delineates
in the pancreatic epithelium a subset of cells, corresponding to
endocrine and ductal precursors, by inducing the expression of
HNF-1β [112]. Finally, HNF-6 stimulates the differentiation
of endocrine precursors, via induction of the transcription factor
Ngn-3 [113]. Type II diabetic patients were screened for mutations
in the HNF6 gene, but none have been identified so far. Hnf6
c 2004 Biochemical Society
knockout mice also lack a gall bladder and their extrahepatic
bile ducts are abnormal. Their liver is cholestatic and displays
anomalies that resemble human biliary diseases called ‘ductal
plate malformations’. Indeed, the differentiation of cholangiocytes and morphogenesis of the intrahepatic bile ducts are perturbed. These disorders involve the transcription factor HNF-1β,
which is a target of HNF-6 [114]. Recent data from promoter
microarrays following chromatin immunoprecipitation suggest
that HNF-6 serves as a master regulator for feedforward loops in
liver and pancreas [115]. As OC-2 and OC-3 are also expressed
in midgut endoderm, the discovery of the OC class of transcription
opens exciting perspectives in the field of developmental biology.
PFKFB2 gene
The 5 flanking sequence of PFKFB2 contains regions that are
conserved between the human, bovine and rat genes. One finds
in these regions potential binding sites for the Sp1, HNF-1 and
βHLH (helix–loop–helix) (E boxes) transcription factors and for
the GR [20,22,23], but a factor binding to these sites has not been
reported.
PFKFB3 gene
Expression of the ubiquitous isoform is induced by progestins
[26] and by insulin [116]. Potential binding sites for the progesterone receptor and for SREBP (sterol-regulatory-element-binding protein)-1c, a transcription factor known to mediate insulin
action, are present in the 5 flanking sequence of the gene, which
also contains consensus sequences for binding the ubiquitous
factors Sp-1, AP-2 (activating protein 2), and NF-1 [15].
The concentration of the mRNA coding for the inducible isoform increases in human monocytes when they are exposed to
lipopolysaccharide. This could result from increased stability of
the mRNA, whose 3 untranslated region contains typical AUUUA
regulatory sequences [27]. Transcription of the PFKFB3 gene also
increases following PKC (protein kinase C) and PKA stimulation
[15] and in response to hypoxia, an effect that requires the
HIF-1 (hypoxia-inducible transcription factor-1) [117]. However,
these data were obtained with mRNA probes that do not allow
distinction between the ubiquitous and the inducible isoforms.
MECHANISMS OF CONTROL OF PFK-2 AND FBPase-2
BY PROTEIN PHOSPHORYLATION
Although protein phosphorylation has been recognized for more
than 40 years as the main mechanism for controlling enzyme
activity and protein function, we know surprisingly little about
how the introduction of a phosphate group on specific serine,
threonine and tyrosine residues leads to its biological effects.
Historically, the first 3D structures revealing mechanisms of phosphorylation-induced changes in enzyme activity came from those
of rabbit muscle glycogen phosphorylase [118] and Escherichia
coli isocitrate dehydrogenase [119]. Crystal structures of phosphorylase in its phosphorylated and dephosphorylated forms
showed that the phosphorylation of Ser14 at the subunit interface
induces the N-terminus to adopt a α-helical conformation. This
change is transmitted to the active site some 30 Å (1 Å = 0.1 nm)
away and results in the T-to-R transition [118]. On the other hand,
phosphorylation of E. coli isocitrate dehydrogenase at Ser113 inactivates the enzyme not via a conformational change [119], but
by charge repulsion between the phosphorylated serine and the
negatively charged substrate [120]. Phosphorylation of protein
kinases, such as the insulin receptor kinase or the AGC kinases in
the so-called ‘activation loop’, leads to activation by electrostatic
interaction between the negatively charged phosphate group and
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
positively charged residues in the upper lobe, which stabilizes the
closed conformation [121]. Both conformational changes and/or
direct electrostatic effects in the substrate-binding sites could be
responsible for the control of PFK-2 and FBPase-2 activities by
phosphorylation.
Rat liver PFK-2/FBPase-2
Although the human liver isoenzyme has recently been crystallized for 3D structural analysis, the N-terminus was not solved
[12], possibly because of its high flexibility. Phosphorylation
of the liver isoenzyme at Ser32 by PKA [122] leads to PFK-2
inactivation, mainly by increasing the K m for Fru-6-P [123–125].
Therefore movement of the phosphorylated Ser32 into the PFK-2
domain, where it could repel the negatively charged phosphate
group of the substrate, might explain the increase in K m for
Fru-6-P. At the same time, a conformational change transmitted
to the FBPase-2 domain would explain how phosphorylation
of Ser32 by PKA activates FBPase-2 by increasing the V max
[124,126,127]. Mutation of Ser32 to aspartic acid in the rat liver
isoenzyme [128], or introduction of a phosphorylation site motif
in the N-terminus of the testis isoenzyme and its subsequent
phosphorylation by PKA [129], inactivated PFK-2 and activated
FBPase-2 as expected.
FBPase-2 activity seems to be repressed by the PFK-2 domain,
since partial proteolysis of the liver isoenzyme, which removes
most of the PFK-2 domain, leads to a 2-fold increase in V max of
FBPase-2 [130]. Regulation of the PFK-2 and FBPase-2 activities
by PKA-induced phosphorylation appears to involve both the Nand C-termini. Deletion of the N-terminal 22 residues of the liver
isoenzyme increased the kcat of FBPase-2 4-fold and decreased
the affinity of PFK-2 for Fru-6-P 20-fold [131]. This deletion
also blunted the PKA-induced effects of phosphorylation on both
activities. Truncation of the C-terminal 25–30 residues from the
rat liver [132] and chicken liver [133] isoenzymes increased the kcat
of FBPase-2 and abolished the effect of PKA-mediated phosphorylation to activate FBPase-2. A sequence His444 -Arg-Glu-Arg447
was identified in chicken liver PFK-2/FBPase-2, which mediates the repressive effect of the N-terminal PFK-2 domain on
FBPase-2 activity [133]. It was proposed that phosphorylation by
PKA could activate FBPase-2 by relieving the repression by the
PFK-2 domain that is mediated by the two basic residues, His444
and Arg445 , in this sequence [133]. However, it is unlikely that
this His-Arg-Glu-Arg sequence interacts directly with the N-terminus or could form the serine-phosphate recognition site, since,
assuming the chicken liver bifunctional enzyme structure is the
same as that of the testis isoenzyme, the N-terminus would be
located too far away (some 50 Å). In the rat liver isoenzyme, this
motif corresponds to His446 -Arg-Asp-Lys449 and in the testis isoenzyme to His444 -Arg-Asp-Arg447 , which in the crystal structure
would be located in the FBPase-2 domain. As in glycogen phosphorylase, phosphorylation could induce a long-range conformational change by introducing new charge–charge interactions
resulting in a more catalytically competent active site.
Bovine heart PFK-2/FBPase-2
The mechanism of control of PFK-2 by phosphorylation of the
heart isoenzyme is also difficult to explain in the absence of a
crystal structure of the phosphorylated isoenzyme. Phosphorylation of Ser466 and Ser483 at the C-terminal end of the bovine heart
isoenzyme by PKA [134–137] and insulin-stimulated protein kinases [137] activates PFK-2 by decreasing the K m for Fru-6-P
and by increasing the V max without affecting FBPase-2. Mutagenesis of Ser466 to glutamic acid and Ser483 to glutamic acid, to
mimic the effects of phosphorylation, indicated that Ser466 phos-
571
phorylation is responsible for the increase in V max , whereas both
Ser466 and Ser483 phosphorylation are necessary to decrease the K m
for Fru-6-P [138]. The C-terminal ten residues in the 3D structure of the rat testis isoenzyme are indicated by arrows in Figure 4(A). In the bovine heart isoenzyme, the C-terminus is 60
residues longer and could conceivably stretch into the PFK-2
domain. The role of the C-terminus in the bovine heart isoenzyme
was demonstrated by deletion mutagenesis. Deletion of the 49
C-terminal residues, removing Ser483 , but leaving the sequence
around Ser466 intact, led to a slight increase in V max , slightly
decreased the K m for Fru-6-P and increased the kcat /K m of PFK-2
2-fold without affecting FBPase-2 [139]. Furthermore, the V max
of PFK-2 of the truncated enzyme was increased following phosphorylation by PKA. Deletion of 78 C-terminal residues, which
removes both PKA phosphorylation sites, led to a 2.5-fold
increase in V max , a 4.5-fold decrease in K m for Fru-6-P and
12-fold increased kcat /K m of PFK-2 [139]. This longer deletion
also activated FBPase-2 through a 3-fold increase in kcat /K m . A
possible interpretation is that the C-terminal end represses the
PFK-2 and FBPase-2 domains and that phosphorylation by PKA
at Ser466 and Ser483 relieves the inhibition of PFK-2 either by displacing the C-terminus from the PFK-2 domain or via the transmission of conformational changes.
PHOSPHORYLATION OF PFK-2/FBPase-2 ISOENZYMES
AND THE CONTROL OF GLYCOLYSIS
Lower eukaryotes
In yeast incubated with high concentrations of glucose, cAMP
concentrations rise, PKA is stimulated and then phosphorylates
and activates PFK-2 [82]. When the yeast pfk26 isoform was
cloned and sequenced, it was suggested that phosphorylation at
Ser644 in the C-terminal sequence Arg-Arg-Tyr-Ser would be responsible for PFK-2 activation [83], by analogy with the heart isoenzyme. However, the yeast pfk26 Ser644 → Ala mutant was still
phosphorylated in vitro by PKA or in vivo in response to glucose,
and a second PKA site was suggested as Thr157 [140]. In vitro, both
Ser644 and Thr157 were phosphorylated by PKA; however, in vivo,
only Ser644 phosphorylation was the result of PKA stimulation
[141]. In yeast, lowering glucose concentrations activates Snf1
kinase, the homologue of AMPK (AMP-activated protein kinase)
in mammals [142]. AMPK phosphorylates and activates heart
PFK-2 and was suggested to be involved in the Pasteur effect in
this tissue (see below). However, it is unlikely that Snf1 would
be involved in the regulation of glycolysis in yeast in response
to glucose, unless phosphorylation at a site different from the
PKA site would inactivate PFK-2. Under hypotonic conditions,
yeast PFK-2 becomes inactivated via phosphorylation at Ser652 ,
probably by yeast PKC1 [143].
In plants, Fru-2,6-P2 levels change diurnally and the targets of
Fru-2,6-P2 are the pyrophosphate:fructose-6-phosphate 1-phosphotransferase, an alternative PFK-1 homologue, and FBPase-1
[144,145]. Low Fru-2,6-P2 concentrations favour sucrose synthesis from photosynthetically assimilated carbon, whereas
increased Fru-2,6-P2 concentrations promote starch synthesis. In
A. thaliana, a principally serine-phosphorylated form of PFK-2/
FBPase-2 has been detected [146]. However, the protein kinase(s)
were not identified, and although the phosphorylated form was
predominant at the beginning of the dark period, it is not known
whether phosphorylation would result in changes in PFK-2 or
FBPase-2 activity.
PFK-2 has been purified to homogeneity from the hepatopancreas of an anoxia-tolerant marine whelk as an M r 67 000 subunit dimer with negligible FBPase-2 activity [147]. Anoxia
c 2004 Biochemical Society
572
M. H. Rider and others
depressed Fru-2,6-P2 concentrations and PFK-2 activity in all
organs of this mollusc via a phosphorylation mechanism, but
PKA or PKC were not involved. An intriguing possibility is that
AMPK might be implicated; however, if so, this would have the
opposite effect on PFK-2 activity as that described for the heart
enzyme (see below).
Mammals
The liver and muscle isoenzymes
In the liver, glucagon stimulates PKA, which phosphorylates
Ser32 of the liver isoenzyme, leading to PFK-2 inactivation and
FBPase-2 activation (reviewed in [1–7]). The subsequent fall in
Fru-2,6-P2 concentration decreases glycolysis and increases gluconeogenic flux. Together with the PKA-induced phosphorylation
and inactivation of pyruvate kinase, this is the basis of the effect
of glucagon to block hepatic glycolysis and stimulate gluconeogenesis during starvation. Apart from control by PKA, no other
meaningful phosphorylations of the liver isoenzyme have been
described. Indeed, in intact hepatocytes, PFK-2/FBPase-2 was
phosphorylated by glucagon, but not by treatment with vasopressin, phorbol ester or calcium ionophore [148]. In contrast,
an increase in Fru-2,6-P2 was observed in hepatocytes incubated
with vasopressin, probably as a result of an increase in glycogen
breakdown [149].
Insulin stimulates glycolysis in white adipose tissue and skeletal muscle. The muscle isoenzyme is a splice variant of the
liver isoenzyme with a different N-terminus, where the first 32 residues, including the PKA phosphorylation site, are replaced by
a peptide devoid of phosphorylation sites (see above). White adipose tissue [19] and skeletal muscle [150] contain both the liver
and muscle isoenzymes. In skeletal muscle, Fru-2,6-P2 concentrations are increased by insulin [151], adrenaline [151–153] and
contraction induced by exercise [154,155] or electrical stimulation
[152,156,157], probably secondary to a rise in Fru-6-P. In white
adipose tissue, insulin stimulates glycolysis without increasing
Fru-2,6-P2 concentrations or affecting PFK-2 activity [158]. The
stimulation of glycolysis by insulin in white adipose tissue and
skeletal muscle is thus likely to be mainly due to the well-known
stimulation of glucose transport [159].
The heart isoenzyme
Insulin stimulates glycolysis in heart by a combination of an increase in glucose transport and the activation of PFK-2 [8,9]. Rat
heart PFK-2 is activated by insulin in vivo through a 2-fold increase in V max , with no change in K m for Fru-6-P [160]. The heart
can oxidize different fuels to sustain contraction. Lipid fuels (fatty
acids and ketone bodies) are preferentially used, which helps to
conserve glucose in the fasting state. However, when blood glucose concentrations rise following a meal, the subsequent rise in
insulin would allow the heart to switch to glucose utilization by
stimulating glycolysis [8]. It is not clear why heart, and not adipose
tissue and skeletal muscle, should have evolved with an insulinactivated PFK-2. A possible explanation is that following insulin-stimulated glucose transport, glucose 6-phosphate is directed
towards glycogen in skeletal muscle [161], whereas in heart, glucose 6-phosphate would be mainly routed towards pyruvate [8].
An interesting parallel can be drawn with acetyl-CoA carboxylase (ACC), where an isoform is present in heart and skeletal
muscle (ACC2) [162] that is different from the liver and adipose tissue isoenzyme (ACC1) [163]. Heart and skeletal muscle
have little lipogenic capacity and the role of ACC2 is probably just
to control fatty acid oxidation through changes in malonyl-CoA
concentrations [164,165].
c 2004 Biochemical Society
Considerable advances towards the understanding of insulin
action [166–169] have been made since we first described heart
PFK-2 activation by insulin many years ago [160]. There are
several signalling pathways which can be triggered following the
binding of insulin to its receptor. Insulin receptor binding leads to
autophosphorylation and activation of its receptor kinase, which
in turn phosphorylates members of the IRS family, Shc and Cbl
[169]. Cbl phosphorylation in lipid rafts has been proposed to be
involved in GLUT4 (glucose transporter 4) vesicle translocation
to the plasma membrane. The adaptor proteins Grb2 and Sos
lead to activation of the MAPK cascade via Ras stimulation, while
stimulation of the PI3K pathway leads to the activation of p70S6k
(p70 ribosomal protein S6 kinase) and PKB [167,168]. PKB is
activated in response to insulin via phosphorylation in the activation loop at Thr308 by PDK (phosphoinositide-dependent protein
kinase)-1 and by phosphorylation in the hydrophobic motif at
Ser473 by PDK2. The involvement of the insulin signalling cascades can be studied by the careful use of inhibitors. In rat cardiomyocytes, we showed that the insulin-induced activation of PFK-2
was blocked by wortmannin, a PI3K inhibitor, but was insensitive
to rapamycin or PD098059, which prevent the activation of p70S6k
and the MAPK cascade respectively [170]. These experiments
with inhibitors suggested that PI3K, but not p70S6k , is involved in
the activation of PFK-2 in response to insulin. In vitro experiments
with purified enzyme preparations showed that PKB phosphorylated and activated recombinant heart PFK-2 [137]. The phosphorylation sites were identified as Ser466 and Ser483 in the bovine
heart isoenzyme. Interestingly, 14-3-3 proteins bind to phosphorylated Ser483 and this might contribute towards PFK-2 activation
[171]. To understand further the mechanism for the activation
of heart PFK-2 by insulin, HEK-293 (human embryonic kidney)
cells were transfected with protein kinase constructs. In cells overexpressing heart PFK-2, insulin activated PFK-2 by phosphorylation of Ser466 and Ser483 [138]. Co-transfection with dominantnegative PDK1 abolished PFK-2 activation by insulin. However,
transfection of dominant-negative PKB failed to prevent insulininduced PFK-2 activation [138]. Therefore PDK1 appears to
be necessary for the insulin-induced activation of heart PFK-2,
whereas PKB is not essential. This was supported further by
the failure of insulin to activate heart PFK-2 in vivo in PDK1deficient mice [172]. Purification of insulin-stimulated PFK-2s
from perfused hearts revealed the presence of a wortmanninsensitive, insulin-stimulated protein kinase, which we called
‘WISK’ and which did not correspond to PKB or PDK1 [173].
The nature of this protein kinase is currently under investigation.
Bovine heart PFK-2 is also a substrate of PKC [10,134,135],
which phosphorylates Ser84 , Ser466 and Ser475 [135,136]. The preparations of PKC used in these early experiments were from rat
brain and were not separated into the various PKC isoforms.
Therefore these preparations were probably mixtures of ‘conventional’ PKC isoforms, i.e. principally PKCα, PKCβ and PKCγ
[174]. Although PKC phosphorylated sites in the C-terminal
domain (Ser466 and Thr475 ), phosphorylation did not lead to a
change in PFK-2 activity [10,135]. This might be due to the fact
that phosphorylation of a third site in the PFK-2 domain (Ser84 )
would counteract the effects of phosphorylation at the activating
C-tail sites. Site-directed mutagenesis of the PKC sites to glutamic
acid/aspartic acid will be required to solve this issue. At present,
therefore, the physiological significance of the phosphorylation
of heart PFK-2 by PKCs is unclear, although distinct isoforms,
such as members of the ‘atypical’ PKCs, might phosphorylate
Ser466 specifically and activate PFK-2. This could be relevant to
the activation of heart PFK-2 by insulin, as the WISK preparation
might contain an atypical PKC isoform such as PKCζ (V. Mouton,
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
Figure 7 Conserved C-terminal domains in the heart and brain/placenta
PFK-2/FBPase-2 isoenzymes
The C-terminal sequence of human heart PFK-2/FBPase-2 containing the phosphorylation sites,
Ser466 and Ser483 , was aligned by eye with C-terminal sequences of the heart isoenzyme from
other species and with the brain/placenta isoenzyme. The residue corresponding to Ser466 is in a
suitable consensus for phosphorylation by AMPK in all sequences, whereas the brain/placenta
isoenzyme lacks the − 5 arginine residue for phosphorylation by insulin-stimulated protein
kinases, although phosphorylation by PKA and PKC would still be expected (see text).
D. Vertommen, L. Bertrand, L. Hue and M. H. Rider, unpublished
work).
Adrenaline, a positive inotropic hormone, increases Fru-2,6-P2
concentrations and stimulates glycolysis in heart [175]. The phosphorylation and activation of heart PFK-2 by PKA could participate in this effect. Indeed, phosphorylation of heart PFK-2 by
PKA in vitro activated PFK-2 by virtue of an increase in V max and
a decrease in K m for Fru-6-P [134,135,137]. Moreover, the phosphorylation sites were identified as Ser466 and Ser483 in the bovine
heart isoenzyme [136], i.e. the same sites phosphorylated by PKB
and other insulin-stimulated protein kinases [137]. However, the
involvement of PKA in the stimulation of heart glycolysis by
adrenaline is questionable, because the activation of PFK-2 is
difficult to demonstrate (C. Beauloye, L. Bertrand, M. H. Rider
and L. Hue, unpublished work). The rise in Fru-2,6-P2 might be
secondary to an increase in Fru-6-P concentration [175]; however,
such an increase has not always been observed [152].
Heart glycolysis is also stimulated when the oxygen supply
is restricted, as in myocardial ischaemia or anoxia (the Pasteur
Figure 8
573
effect). Under these conditions, the only source of ATP is glycolysis, and thus the only substrates the heart can use are glucose and
glycogen. Anaerobic conditions cause ATP concentrations to fall
and AMP rises in parallel. These changes in adenine nucleotide
levels lead to AMPK activation [142], which stimulates glycolysis
by increasing GLUT4 translocation [176] and by activating heart
PFK-2 [177]. This contrasts with the situation in liver where
Fru-2,6-P2 concentrations decrease during anoxia [178]. Heart
PFK-2 is a good substrate for AMPK, and phosphorylation leads
to an increase in V max with no change in K m for Fru-6-P. The phosphorylation site was identified as Ser466 [177]. This observation
is consistent with the presence of hydrophobic residues at − 4 and
+ 4 relative to the phosphorylated serine residue. In addition, there
are arginine residues at − 3 and − 2 for phosphorylation by PKA
and arginine residues at − 5 and − 3 plus a hydrophobic residue
at + 1 for recognition by insulin-stimulated protein kinases
(Figure 7).
Glycolysis in heart also increases in response to increasing the
workload [179,180]. Under these conditions, Fru-2,6-P2 concentrations rise and heart PFK-2 is activated. We originally proposed
that phosphorylation and activation of heart PFK-2 by Ca/CAMK
(Ca2+ /calmodulin-activated protein kinase), secondary to a rise
in cytoplasmic Ca2+ , could explain this effect [179]. However,
recent studies from our laboratory indicate that this might not be
the whole story. Surprisingly, submitting hearts to an increased
workload did not activate AMPK [180]. However, increasing
the workload did activate PKB, but not p70S6k . Furthermore, the
activation of PKB and PFK-2 due to increasing the workload was
blocked by wortmannin, but was rapamycin-insensitive [180].
Therefore a possible scenario is that PFK-2 activation could be due
to shear-stress-induced PI3K activation and activation of PKB.
A summary of the various protein kinases that converge on the
phosphorylation of heart PFK-2 is depicted in Figure 8.
Brain/placenta isoenzyme
Another PFK-2/FBPase-2 that contains the equivalent of Ser466 in
a suitable motif for AMPK is the brain/placenta isoenzyme with its
Potential protein kinase cascades converging on heart PFK-2/FBPase-2
The numbering of residues refers to the bovine heart isoenzyme. The signalling pathways leading to Ser466 phosphorylation of the heart isoenzyme could also impinge on Ser461 of the brain/placenta
isoenzyme, but not on the equivalents of Ser84 or Thr475 , which are replaced by non-phosphorylatable residues in the brain/placenta isoenzyme.
c 2004 Biochemical Society
574
M. H. Rider and others
‘ubiquitous’ and ‘inducible’ (iPFK-2, see above) isoforms (Figure 7). In iPFK-2, Ser461 corresponds to Ser466 and phosphorylation
by AMPK activates PFK-2, as expected [181]. The expression of
iPFK-2 is induced in monocytes exposed to lipopolysaccharide,
upon which it can be phosphorylated and activated by AMPK
[181]. The stimulation of monocyte glycolysis via the AMPK-induced phosphorylation and activation of iPFK-2 could be important for furnishing ATP to sustain cytokine synthesis in infected
anaerobic tissues. iPFK-2 is also expressed constitutively in several cancer cell lines [27], which display high rates of glycolysis,
even in the presence of oxygen (Warburg effect). Since tumours
contain anoxic zones, the activation of AMPK in cancer cells
in these areas could stimulate glycolysis further by phosphorylating iPFK-2. It would be interesting to see whether insulin
could activate the brain/placenta PFK-2 isoenzyme, in which the
consensus around Ser461 is similar to that around Ser466 phosphorylated in response to insulin in the heart isoenzyme (Figure 7).
Indeed, Ser461 of the brain/placenta isoenzyme is phosphorylated
by PKA, PKC [182] and AMPK [181] like Ser466 in heart PFK-2/
FBPase-2 [137,178]. The major PFK-2/FBPase-2 isoform in
astrocytes is iPFK-2 [183]. In these cells, but not in neurons, NO
stimulated glycolysis and increased Fru-2,6-P2 concentrations.
This effect was attributed to mitochondrial respiratory chain
inhibition at cytochrome oxidase by NO, activation of AMPK
and phosphorylation and activation of iPFK-2 [183]. The resulting
cytoprotective effect of stimulating glycolysis by NO in astrocytes
was absent in neurons, where iPFK-2 protein was undetectable.
Isoenzymes in proliferating cells
PFK-2/FBPase-2 present in various cells and cell lines including
cancer cells [27,184–187], foetal liver [188], regenerating liver
[189] and lymphocytes [190] has properties similar to those of the
heart and brain/placenta isoenzymes. Foetal liver was subsequently shown to contain equal amounts of the L and M mRNAs [191]
and regenerating liver was shown to contain the F mRNA
[192]. Fru-2,6-P2 concentrations and PFK-2 activity were increased in chick embryo fibroblasts upon transformation by retroviruses carrying the v-src oncogene [193]. The oncogene-dependent transformation seems to involve PKC and both RNA and
protein synthesis. Therefore pp60 v-src, acting via PKC, could
change the isoenzyme expression of PFK-2/FBPase-2 [194].
Phorbol esters increase Fru-2,6-P2 concentrations in chick embryo
fibroblasts [195], in human fibroblasts [196], in human platelets
[197], in differentiating megakaryocytes [198], in human B-lymphocytes [199], in mouse spleen lymphocytes and purified B-cells
[190], and in foetal rat hepatocytes [200]. The effect of phorbol
esters to increase Fru-2,6-P2 in these systems could be attributed to
a rise in hexose 6-phosphates and in some cases to PFK-2 activation. In foetal liver, although PFK-2/FBPase-2 was phosphorylated by PKC, the activity of PFK-2 was unaffected [188], as
described above for the heart isoenzyme. Therefore, in foetal
hepatocytes, phorbol esters probably do not activate PFK-2 via
PKC-induced phosphorylation. In chick embryo fibroblasts, the
activation of PFK-2 by phorbol esters involves the increased synthesis of an isoenzyme of PFK-2/FBPase-2, which might be
identical with the heart isoenzyme or iPFK-2 (see above). Supposing that the heart or brain/placenta isoenzyme is expressed in
these systems, PFK-2 activation via phorbol ester/PKC activation
might be secondary to the activation of the MAPK cascade [201]
and phosphorylation by MAPKAPK-1 (MAPK-activated protein
kinase-1) [137]. The increase in Fru-2,6-P2 concentration and
PFK-2 activity in thrombin-stimulated platelets [197], and in
fibroblasts stimulated by growth factors [196] might also be mediated by this pathway. In A431 cells treated with EGF, the increase
c 2004 Biochemical Society
in Fru-2,6-P2 and PFK-2 activity was not due to an increase in
transcription or translation [202]. Furthermore, PKC did not appear to be involved in the signalling pathway, and a role for Ca2+ ,
possibly acting via the multifunctional Ca/CAMK, was postulated
[202]. Lastly, insulin has been shown to increase Fru-2,6-P2 concentrations and to activate PFK-2 in several cultured cells, including human fibroblasts [203], chick embryo fibroblasts [195],
chick embryo hepatocytes [204] and foetal rat hepatocytes [200].
These effects could be mediated by a novel protein kinase of
the insulin signalling cascades, as described above for the heart
isoenzyme.
CONCLUDING REMARKS
The 3D structure of PFK-2/FBPase-2 revealed that the PFK-2
domains in the homodimer come together in a head-to-head
fashion with the FBPase-2 domains being almost independent.
Differences in the PFK-2/FBPase-2 activity ratio between the
mammalian isoenzymes could be explained by differences in
flexibility at the PFK-2/FBPase-2 domain interface and interactions with the different N- and C-termini and the catalytic sites.
The PFK-2 domain has a similar fold to AK and is not related to
PFK-1, as originally proposed.
The presence of a PFK-2/FBPase-2 sequence in Desulfovibrio
raises the possibility that it was horizontally transferred from
a unicellular eukaryote. Gene fusion between the PFK-2 and
FBPase-2 catalytic domains must have occurred early in eukaryotic evolution, in the common ancestor of this gene donor, the
trypanosomatids and all other eukaryotes. In trypanosomatids,
monofunctional isoenzymes have arisen in which either the
PFK-2 or the FBPase-2 domain has become inactive. These isoenzymes might become expressed at different stages of the parasite’s
life cycle to enable the organism to adjust to different environments. It will be interesting to see if ‘primitive’ protist lineages
can be found in which monofunctional, single domain PFK-2 and
FBPase-2 enzymes occur or in which no traces of these enzymes
and Fru-2,6-P2 can be found.
Studies on both the long- and short-term control of the PFK-2/
FBPase-2 system have proved to be fruitful areas of research,
leading to the identification of protein kinase cascades that converge on the heart and brain/placenta isoenzymes and of the novel
transcription factor, HNF-6. Investigations on the mechanism of
heart PFK-2 activation by insulin could result in the recognition
of new elements in insulin signalling to metabolic pathways that
may not necessarily be mediated by PKB. Studies on the phosphorylation of the heart and brain/placenta isoenzymes by AMPK
have provided new explanations for the old observations of the
Pasteur and Warburg effects in terms of a protein phosphorylation
mechanism. It is likely that other protein kinases will be found
that impinge on the heart and brain/placenta isoenzymes. Good
candidates are members of the AGC family, e.g. protein kinase G and the serum- and glucocorticoid-activated protein
kinases. Clearly, more in vivo studies will be needed to assess the
physiological relevance of heart and brain/placenta PFK-2 phosphorylation.
Interesting new avenues of research are isoenzyme overexpression and tissue-specific gene knockouts. Overexpression of
PFK-2/FBPase-2 in mouse liver has been shown to lower blood
glucose levels by increasing Fru-2,6-P2 and lowering hepatic glucose production [205]. Another field of interest is control by protein–protein interactions. The liver and brain/placenta isoenzymes
were shown to bind to glucokinase [206]. This could be involved
in channelling glycolytic intermediates. In pancreatic islets, the
interaction between PFK-2/FBPase-2 and glucokinase stimulates
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
glucose phosphorylation and its subsequent metabolism, which
could be involved in glucose-induced insulin secretion [207].
We thank Professor F. R. Opperdoes [Institute of Cellular Pathology (ICP)/Université
Catholique de Louvain (UCL), Brussels] for help with the phylogenetic analysis and
Professor E. Van Schaftingen (ICP/UCL, Brussels) for pointing out the Desulfovibrio
sequence. Work in the authors’ laboratory was supported by grants from the Interuniversity
Attraction Poles Program – Belgian Science Policy, by the Directorate General Higher
Education and Scientific Research, French Community of Belgium, by the Fund for
Medical Scientific Research (Belgium) and by EU contract no. QLG1-CT-2001-01488
(AMPDIAMET).
REFERENCES
1 Van Schaftingen, E. (1987) Fructose 2,6-bisphosphate. Adv. Enzymol. 59, 315–395
2 Hue, L. and Rider, M. H. (1987) Role of fructose 2,6-bisphosphate in the control of
glycolysis in mammalian tissues. Biochem. J. 245, 313–324
3 Pilkis, S. J., El-Maghrabi, M. R. and Claus, T. H. (1988) Hormonal regulation of hepatic
gluconeogenesis and glycolysis. Annu. Rev. Biochem. 57, 755–783
4 Hue, L., Rider, M. H. and Rousseau, G. G. (1990) Fructose-2,6-bisphosphate in extra
hepatic tissues. In Fructose-2,6-bisphosphate (Pilkis, S. J., ed.), pp. 173–193,
CRC Press, Boca Raton
5 Van Schaftingen, E., Mertens, E. and Opperdoes, F. R. (1990) Fructose-2,6bisphosphate in primitive systems. In Fructose-2,6-bisphosphate (Pilkis, S. J., ed.),
pp. 229–244, CRC Press, Boca Raton
6 Pilkis, S. J., Claus, T. H., Kurland, I. J. and Lange, A. J. (1995) 6-Phosphofructo2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme.
Annu. Rev. Biochem. 64, 799–835
7 Okar, D. A., Manzano, A., Navarro-Sabate, A., Riera, L., Bartrons, R. and Lange, A. J.
(2001) PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6bisphosphate. Trends Biochem. Sci. 26, 30–35
8 Depré, C., Rider, M. H. and Hue, L. (1998) Mechanisms of control of heart glycolysis.
Eur. J. Biochem. 258, 277–290
9 Hue, L., Beauloye, C., Marsin, A.-S., Bertrand, L., Horman, S. and Rider, M. H. (2002)
Insulin and ischaemia stimulate glycolysis by acting on the same targets through
different and opposing signaling pathways. J. Mol. Cell. Cardiol. 34, 1091–1097
10 Rider, M. H. and Hue, L. (1986) Phosphorylation of purified bovine heart and rat liver
6-phosphofructo-2-kinase by protein kinase C and comparison of the fructose-2,6bisphosphatase activity of the two enzymes. Biochem. J. 240, 57–61
11 Sakata, J., Abe, Y. and Uyeda, K. (1991) Molecular cloning of the DNA and expression
and characterization of rat testis fructose-6-phosphate,2-kinase:fructose-2,6bisphosphatase. J. Biol. Chem. 266, 15764–15770
12 Lee, H.-T., Li, Y., Uyeda, K. and Hasemann, C. A. (2002) Tissue-specific structure/
function differentiation of the liver isoform of 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase. J. Biol. Chem. 278, 523–530
13 Darville, M. I., Crepin, K. M., Hue, L. and Rousseau, G. G. (1989) 5 flanking sequence
and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase. Proc. Natl. Acad. Sci. U.S.A. 86, 6543–6547
14 Darville, M. I., Chikri, M., Lebeau, E., Hue, L. and Rousseau, G. G. (1991) A rat gene
encoding heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. FEBS Lett.
288, 91–94
15 Navarro-Sabate, A., Manzano, A., Riera, L., Rosa, J. L., Ventura, F. and Bartrons, R.
(2001) The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
gene (PFKFB3 ): promoter characterization and genomic structure. Gene 264, 131–138
16 Manzano, A., Perez, J. X., Nadal, M., Estivill, X., Lange, A. and Bartrons, R. (1999)
Cloning, expression and chromosomal localization of a human testis 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase gene. Gene 229, 83–89
17 Darville, M. I., Antoine, I. V. and Rousseau, G. G. (1992) Characterization of an enhancer
upstream from the muscle-type promoter of a gene encoding 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase. Nucleic Acids Res. 20, 3575–3583
18 Dupriez, V. J., Darville, M. I., Antoine, I. V., Gegonne, A., Ghysdael, J. and Rousseau,
G. G. (1993) Characterization of a hepatoma mRNA transcribed from a third promoter of a
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-encoding gene and controlled
by ets oncogene-related products. Proc. Natl. Acad. Sci. U.S.A. 90, 8224–8228
19 Bruni, P., Vandoolaeghe, P., Rousseau, G. G., Hue, L. and Rider, M. H. (1999) Expression
and regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes in
white adipose tissue. Eur. J. Biochem. 259, 756–761
20 Chikri, M. and Rousseau, G. G. (1995) Rat gene coding for heart 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase: characterization of an unusual promoter region
and identification of four mRNAs. Biochemistry 34, 8876–8884
575
21 Vidal, H., Crepin, K. M., Rider, M. H., Hue, L. and Rousseau, G. G. (1993) Cloning and
expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
from bovine heart. FEBS Lett. 330, 329–333
22 Tsuchiya, Y. and Uyeda, K. (1994) Bovine heart fructose 6-P,2-kinase:fructose
2,6-bisphosphatase mRNA and gene structure. Arch. Biochem. Biophys. 310, 467–474
23 Heine-Suner, D., Diaz-Guillen, M. A., Lange, A. J. and Rodriguez de Cordoba, S. (1998)
Sequence and structure of the human 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase heart isoform gene (PFKFB2 ). Eur. J. Biochem. 254, 103–110
24 Ventura, F., Ambrosio, S., Bartrons, R., El-Maghrabi, M. R., Lange, A. J. and Pilkis, S. J.
(1995) Cloning and expression of a catalytic core bovine brain 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase. Biochem. Biophys. Res. Commun. 209,
1140–1148
25 Sakai, A., Kato, M., Fukasawa, M., Ishiguro, M., Furuya, E. and Sakakibara, R. (1996)
Cloning of cDNA encoding for a novel isozyme of fructose 6-phosphate, 2-kinase/
fructose 2,6-bisphosphatase from human placenta. J. Biochem. 119, 506–511
26 Hamilton, J. A., Callaghan, M. J., Sutherland, R. L. and Watts, C. K. (1997) Identification
of PRG1, a novel progestin-responsive gene with sequence homology to
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Mol. Endocrinol. 11, 490–502
27 Chesney, J., Mitchell, R., Benigni, F., Bacher, M., Spiegel, L., Al-Abed, Y., Han, J. H.,
Metz, C. and Bucala, R. (1999) An inducible gene product for 6-phospho-fructo2-kinase with an AU-rich instability element: role in tumor cell glycolysis and the
Warburg effect. Proc. Natl. Acad. Sci. U.S.A. 96, 3047–3052
28 Watanabe, F. and Furuya, E. (1999) Tissue-specific alternative splicing of rat brain
fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase. FEBS Lett. 458, 304–308
29 Kessler, R. and Eschrich, K. (2001) Splice isoforms of ubiquitous 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase in human brain. Mol. Brain Res. 87, 190–195
30 Sakata, J., Abe, Y. and Uyeda, K. (1991) Molecular cloning of the DNA and expression
and characterization of rat testes fructose-6-phosphate,2-kinase:fructose2,6-bisphosphatase. J. Biol. Chem. 266, 15764–15770
31 Kitajima, S., Sakakibara, R. and Uyeda, K. (1984) Kinetic studies of fructose
6-phosphate,2-kinase and fructose 2,6-bisphosphatase. J. Biol. Chem. 259, 6896–6903
32 Kretschmer, M. and Hofmann, E. (1984) Inhibition of rat liver phosphofructokinase-2 by
phosphoenolpyruvate and ADP. Biochem. Biophys. Res. Commun. 124, 793–796
33 Kountz, P. D., Freeman, S., Cook, A. G., El-Maghrabi, M. R., Knowles, J. R. and Pilkis,
S. J. (1988) The stereochemical course of phospho group transfer catalyzed by rat liver
6-phosphofructo-2-kinase. J. Biol. Chem. 263, 16069–16072
34 Bazan, J. F., Fletterick, R. J. and Pilkis, S. J. (1989) Evolution of a bifunctional enzyme:
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Proc. Natl. Acad Sci. U.S.A.
86, 9642–9646
35 Bazan, J. F. and Fletterick, R. J. (1990) Tertiary structure modelling of 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase: structural, functional and evolutionary design of
a bifunctional enzyme. In Fructose-2,6-bisphosphate (Pilkis, S. J., ed.), pp. 125–171,
CRC Press, Boca Raton
36 Crepin, K. M., Vertommen, D., Dom, G., Hue, L. and Rider, M. H. (1993) Rat muscle
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: study of the kinase domain by
site-directed mutagenesis. J. Biol. Chem. 268, 15277–15284
37 Vertommen, D., Bertrand, L., Sontag, B., Di Pietro, A., Louckx, M. P., Vidal, H., Hue, L.
and Rider, M. H. (1996) The ATP binding site in the 2-kinase domain of liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase study of the role of Lys-54 and
Thr-55 by site-directed mutagenesis. J. Biol. Chem. 271, 17875–17880
38 Rider, M. H., Crepin, K. M., De Cloedt, M., Bertrand, L. and Hue, L. (1994) Site-directed
mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase:
role of Asp-130 in the 2-kinase domain. Biochem. J. 300, 111–115
39 Walker, J. E., Saraste, M., Runswick, M. J. and Gay, N. J. (1982) Distantly related
sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other
ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951
40 Traut, T. W. (1994) The functions and consensus motifs of nine types of peptide segments
that form different types of nucleotide-binding sites. Eur. J. Biochem. 222, 9–19
41 Bertrand, L., Vertommen, D., Depiereux, E., Hue, L., Rider, M. H. and Feytmans, E.
(1997) Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase on adenylate kinase. Biochem. J. 321, 615–621
42 Bertrand, L., Vertommen, D., Feytmans, E., Di Pietro, A., Rider, M. H. and Hue, L. (1997)
Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem. J. 321, 609–614
43 Bertrand, L., Deprez, J., Vertommen, D., Di Pietro, A., Hue, L. and Rider, M. H. (1997)
Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem. J. 321, 623–627
44 Bertrand, L., Vertommen, D., Freeman, P. M., Wouters, J., Depiereux, E., Di Pietro, A.,
Hue, L. and Rider, M. H. (1998) Mutagenesis of the fructose 6-phosphate binding site in
the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Eur. J. Biochem. 254, 490–496
c 2004 Biochemical Society
576
M. H. Rider and others
45 Hasemann, C. A., Istvan, E. S., Uyeda, K. and Deisenhofer, J. (1996) The crystal structure
of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
reveals distinct domain homologies. Structure 4, 1017–1029
46 Li, L., Lin, K., Kurland, I. J., Correia, J. J. and Pilkis, S. J. (1992) Site-directed
mutagenesis in rat liver 6-phosphofructo-2-kinase: mutation at the fructose 6-phosphate
binding site affects phosphate activation. J. Biol. Chem. 267, 4386–4393
47 Rider, M. H., Crepin, K. M., De Cloedt, M., Bertrand, L., Vertommen, D. and Hue, L.
(1995) Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis.
Biochem. J. 309, 341–346
48 Tsai, M.-D. and Yan, H. (1991) Mechanism of adenylate kinase: site-directed
mutagenesis versus X-ray and NMR. Biochemistry 30, 6806–6818
49 Frech, M., Darden, T. A., Pederson, L. G., Foley, C. K., Charifson, P. S., Anderson, M. W.
and Wittinghofer, A. (1994) Role of glutamine-61 in the hydrolysis of GTP by p21H-ras:
an experimental and theoretical study. Biochemistry 33, 3237–3244
50 Gideon, P., John, J., Frech, M., Lautwein, A., Clark, R., Scheffer, J. E. and
Wittinghofer, A. (1992) Mutational and kinetic analyses of the GTPase-activating protein
(GAP)–p21 interaction: the C-terminal domain of GAP is not sufficient for full activity.
Mol. Cell. Biol. 12, 2050–2056
51 Kosloff, M. and Selinger, Z. (2001) Substrate-assisted catalysis – application to G
proteins. Trends Biochem. Sci. 26, 161–166
52 Knowles, J. R. (1980) Enzyme-catalyzed phosphoryl transfer reactions.
Annu. Rev. Biochem. 49, 877–919
53 Matte, A., Tari, L. W. and Delbaere, L. T. J. (1998) How do kinases transfer phosphoryl
groups? Structure 6, 413–419
54 Maegley, K. A., Admiraal, S. J. and Hershlag, D. (1996) Ras-catalysed hydrolysis of GTP:
a new perspective from model studies. Proc. Natl. Acad. Sci. U.S.A. 93, 8160–8166
55 Wittinghofer, A. and Pai, E. F. (1991) The structure of Ras protein: a model for a universal
molecular switch. Trends Biochem. Sci. 16, 382–387
56 Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmüller, L., Lautwein, A., Schmitz, F.
and Wittinghofer, A. (1997) The Ras–RasGAP complex: structural basis for GTPase
activation and its loss in oncogenic Ras mutants. Science 277, 333–338
57 Anderson, C. M., Zucker, F. H. and Steitz, T. A. (1979) Space-filling models of kinase
clefts and conformational changes. Science 204, 375–380
58 Schulz, G. E., Müller, C. W. and Diederichs, K. (1990) Induced-fit movements in
adenylate kinases. J. Mol. Biol. 213, 627–630
59 Rider, M. H. and Hue, L. (1992) Inactivation of liver 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase by phenylglyoxal: evidence for essential arginine residues.
Eur. J. Biochem. 285, 405–411
60 Colosia, A. D., Lively, M. O., El-Maghrabi, M. R. and Pilkis, S. J. (1987) Isolation of a
cDNA clone for rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase.
Biochem. Biophys. Res. Commun. 143, 1092–1098
61 Darville, M. I., Crepin, K. M., Vandekerckhove, J., Van Damme, J., Octave, J.-N., Rider,
M. H., Marchand, M. J., Hue, L. and Rousseau, G. G. (1987) Complete nucleotide
sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
derived from a cDNA clone. FEBS Lett. 224, 317–321
62 Algaier, J. and Uyeda, K. (1988) Molecular cloning, sequence analysis, and expression
of a human liver cDNA coding for fructose-6-P,2-kinase: fructose-2,6-bisphosphatase.
Biochem. Biophys. Res. Commun. 153, 328–333
63 Lange, A. J. and Pilkis, S. J. (1990) Sequence of human liver 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase. Nucleic Acids Res. 18, 3652–3652
64 Lange, A. J., El-Maghrabi, M. R. and Pilkis, S. J. (1991) Isolation of bovine liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart
forms of the enzyme are separate gene products. Arch. Biochem. Biophys. 290, 258–263
65 Batra, R. S., Brown, R. M., Brown, G. K. and Craig, I. W. (1996) Molecular cloning and
tissue-specific expression of mouse kidney 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase. FEBS Lett. 393, 167–173
66 Crepin, K. M., Darville, M. I., Michel, A., Hue, L. and Rousseau, G. G. (1989) Cloning
and expression in Escherichia coli of a rat hepatoma cell cDNA coding for
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem. J. 264, 151–160
67 Watanabe, F., Sakai, A., Furuya, E. and Uyeda, K. (1994) Molecular cloning and tissue
specific expression of fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase of rat
brain. Biochem. Biophys. Res. Commun. 198, 335–340
68 Sakata, J. and Uyeda, K. (1990) Bovine heart fructose-6-phosphate 2-kinase/
fructose-2,6-bisphosphatase:complete amino acid sequence and localization of
phosphorylation sites. Proc. Natl. Acad. Sci. U.S.A. 87, 4951–4955
69 Manzano, A., Rosa, J. L., Ventura, F., Perez, J. X., Nadal, M., Estivill, X., Ambrosio, S.,
Gil, J. and Bartrons, R. (1998) Molecular cloning, expression, and chromosomal
localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase gene (PFKFB3 ). Cytogenet. Cell Genet. 83, 214–217
c 2004 Biochemical Society
70 Watanabe, F., Sakai, A. and Furuya, E. (1997) Novel isoforms of rat brain fructose
6-phosphate 2-kinase/fructose 2,6-bisphosphatase are generated by tissue-specific
alternative splicing. J. Neurochem. 69, 1–9
71 Li, L., Lange, A. J. and Pilkis, S. J. (1993) Isolation of a cDNA for chicken liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem. Biophys.
Res. Commun. 190, 397–405
72 Sakai, A., Watanabe, F. and Furuya, E. (1994) Cloning of cDNAs for fructose 6-phosphate
2-kinase/fructose 2,6-bisphosphatase from frog skeletal muscle and liver, and their
expression in skeletal muscle. Biochem. Biophys. Res. Commun. 198, 1099–1106
73 Meton, I., Caseras, A., Mediavilla, D., Fernandez, F. and Baanante, I. V. (1999) Molecular
cloning of a cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
from liver of Sparus aurata : nutritional regulation of enzyme expression.
Biochim. Biophys. Acta 1444, 153–165
74 Villadsen, D., Rung, J. H., Draborg, H. and Nielsen, T. H. (2000) Structure and
hererologous expression of a gene encoding fructose-6-phosphate,2-kinase/
fructose-2,6-bisphosphatase from Arabidopsis thaliana . Biochim. Biophys. Acta 1492,
406–413
75 Draborg, H., Villadsen, D. and Nielsen T. H. (1999) Cloning, characterization and
expression of a bifunctional fructose-6-phosphate, 2-kinase/fructose2,6-bisphosphatase from potato. Plant Mol. Biol. 39, 709–720
76 Markham, J. E. and Kruger, N. J. (2002) Kinetic properties of bifunctional
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.
Eur. J. Biochem. 269, 1267–1277
77 Kretschmer, M., Tempst, P. and Fraenkel, D. G. (1991) Identification and cloning of yeast
phosphofructokinase 2. Eur. J. Biochem. 197, 367–372
78 Boles, E., Gohlmann, H. W. and Zimmermann, F. K. (1996) Cloning of a second gene
encoding 6-phosphofructo-2-kinase in yeast and characterization of mutant strains
without fructose-2,6-bisphosphate. Mol. Microbiol. 20, 65–76
79 Paravicini, G. and Kretschmer, M. (1992) The yeast FBP26 gene codes for a fructose2,6-bisphosphatase. Biochemistry 31, 7126–7133
80 Van Schaftingen, E., Opperdoes, F. R. and Hers, H.-G. (1985) Stimulation of
Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate. Eur. J. Biochem.
153, 403–406
81 Reference deleted
82 François, J., Van Schaftingen, E. and Hers, H.-G. (1984) The mechanism by which
glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces
cerevisiae : a cyclic-AMP-dependent activation of phosphofructokinase 2.
Eur. J. Biochem. 145, 187–193
83 Kretschmer, M. and Fraenkel, D. G. (1991) Yeast 6-phosphofructo-2-kinase: sequence
and mutant. Biochemistry 30, 10663–10672
84 Raamsdonk, L. M., Teusink, B., Broadhurst, D., Zhang, N., Hayes, A., Walsh, M. C.,
Berden, J. A., Brindle, K. M., Kell, D. B., Rowland, J. J. et al. (2001) A functional
genomic strategy that uses metabolome data to reveal the phenotype of silent mutations.
Nat. Biotechnol. 19, 45–50
85 Tauler, A., Lange, A. J., El-Maghrabi, M. R. and Pilkis, S. J. (1989) Expression of rat liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in
Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 86, 7316–7320
86 Tauler, A., Rosenberg, A. H., Colosia, A., Studier, F. W. and Pilkis, S. J. (1988)
Expression of the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase in Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 85,
6642–6646
87 Lange, A. J., Espinet, C., Hall, R., El-Maghrabi, M. R., Vargas, A. M., Miksicek, R. J.,
Granner, D. K. and Pilkis, S. J. (1992) Regulation of gene expression of rat skeletal
muscle/liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: isolation and
characterization of a glucocorticoid response element in the first intron of the gene.
J. Biol. Chem. 267, 15673–15680
88 Zimmermann, P. L. and Rousseau, G. G. (1994) Liver-specific DNase I-hypersensitive
sites and DNA methylation pattern in the promoter region of a 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase gene. Eur. J. Biochem. 220, 183–191
89 Zimmermann, P. L., Pierreux, C. E., Rigaud, G., Rousseau, G. G. and Lemaigre, F. P.
(1997) In vivo protein–DNA interactions on a glucocorticoid response unit of a liverspecific gene: hormone-induced transcription factor binding to constitutively open
chromatin. DNA Cell Biol. 16, 713–723
90 Pierreux, C. E., Urso, B., De Meyts, P., Rousseau, G. G. and Lemaigre, F. P. (1998)
Inhibition by insulin of glucocorticoid-induced gene transcription: involvement of the
ligand-binding domain of the glucocorticoid receptor and independence from the
phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways.
Mol. Endocrinol. 12, 1343–1354
91 De Los Pinos, E., Fernandez De Mattos, S., Joaquin, M. and Tauler, A. (2001) Insulin
inhibits glucocorticoid-stimulated L-type 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase gene expression by activation of the c-Jun N-terminal kinase pathway.
Biochem. J. 353, 267–273
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
92 Darville, M. I., Antoine, I. V., Mertens-Strijthagen, J. R., Dupriez, V. J. and Rousseau,
G. G. (1995) An E2F-dependent late-serum-response promoter in a gene that controls
glycolysis. Oncogene 11, 1509–1517
93 Joaquin, M., Salvado, C., Bellosillo, B., Lange, A. J., Gil, J. and Tauler, A. (1997) Effect
of growth factors on the expression of 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase in Rat-1 fibroblasts. J. Biol. Chem. 272, 2846–2851
94 Fernandez de Mattos, S., De Los Pinos, E. E., Joaquin, M. and Tauler, A. (2000)
Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of
F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role
of protein kinase B and p70 S6 kinase. Biochem. J. 349, 59–65
95 Darville, M. I. and Rousseau, G. G. (1997) E2F-dependent mitogenic stimulation of the
splicing of transcripts from an S phase-regulated gene. Nucleic Acids Res. 25,
2759–2765
96 Dupriez, V. J. and Rousseau, G. G. (1997) Glucose response elements in a gene that
codes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. DNA Cell Biol. 16,
1075–1085
97 Vandoolaeghe, P. and Rousseau, G. G. (1997) C/EBP binds over the TATA box and can
activate the M promoter of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Biochem. Biophys. Res. Commun. 232, 247–250
98 Vandoolaeghe, P., Gueuning, M. A. and Rousseau, G. G. (1999) M-type
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is the product of a late muscle
differentiation gene. Biochem. Biophys. Res. Commun. 259, 250–254
99 Lemaigre, F. P., Durviaux, S. M. and Rousseau, G. G. (1991) Identification of regulatory
sequences and protein-binding sites in the liver-type promoter of a gene encoding
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Mol. Cell. Biol. 11,
1099–1106
100 Lemaigre, F. P., Durviaux, S. M. and Rousseau, G. G. (1993) Liver-specific factor binding
to the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.
J. Biol. Chem. 268, 19896–19905
101 Lemaigre, F. P., Durviaux, S. M., Truong, O., Lannoy, V. J., Hsuan, J. J. and Rousseau,
G. G. (1996) Hepatocyte nuclear factor 6, a transcription factor that contains a novel type
of homeodomain and a single cut domain. Proc. Natl. Acad. Sci. U.S.A. 93, 9460–9464
102 Lannoy, V. J., Burglin, T. R., Rousseau, G. G. and Lemaigre, F. P. (1998) Isoforms of
hepatocyte nuclear factor-6 differ in DNA-binding properties, contain a bifunctional
homeodomain, and define the new ONECUT class of homeodomain proteins.
J. Biol. Chem. 273, 13552–13562
103 Jacquemin, P., Lannoy, V. J., Rousseau, G. G. and Lemaigre, F. P. (1999) OC-2, a novel
mammalian member of the ONECUT class of homeodomain transcription factors whose
function in liver partially overlaps with that of hepatocyte nuclear factor-6. J. Biol. Chem.
274, 2665–2671
104 Vanhorenbeeck, V., Jacquemin, P., Lemaigre, F. P. and Rousseau, G. G. (2002) OC-3, a
novel mammalian member of the ONECUT class of transcription factors.
Biochem. Biophys. Res. Commun. 292, 848–854
105 Nacer-Cherif, H., Bois-Joyeux, B., Rousseau, G. G., Lemaigre, F. P. and Danan, J. L.
(2003) Hepatocyte nuclear factor-6 stimulates transcription of the α-fetoprotein gene
and synergizes with the retinoic acid receptor-related orphan receptor α-4. Biochem. J.
369, 583–591
106 Lahuna, O., Fernandez, L., Karlsson, H., Maiter, D., Lemaigre, F. P., Rousseau, G. G.,
Gustafsson, J. and Mode, A. (1997) Expression of hepatocyte nuclear factor 6 in rat liver
is sex-dependent and regulated by growth hormone. Proc. Natl. Acad. Sci. U.S.A. 94,
12309–12313
107 Pierreux, C. E., Stafford, J., Demonte, D., Scott, D. K., Vandenhaute, J., O’Brien, R. M.,
Granner, D. K., Rousseau, G. G. and Lemaigre, F. P. (1999) Antiglucocorticoid activity of
hepatocyte nuclear factor-6. Proc. Natl. Acad. Sci. U.S.A. 96, 8961–8966
108 Streeper, R. S., Hornbuckle, L. A., Svitek, C. A., Goldman, J. K., Oeser, J. K. and O’Brien,
R. M. (2001) Protein kinase A phosphorylates hepatocyte nuclear factor-6 and
stimulates glucose-6-phosphatase catalytic subunit gene transcription. J. Biol. Chem.
276, 19111–19118
109 Lannoy, V. J., Decaux, J. F., Pierreux, C. E., Lemaigre, F. P. and Rousseau, G. G. (2002)
Liver glucokinase gene expression is controlled by the onecut transcription factor
hepatocyte nuclear factor-6. Diabetologia 45, 1136–1141
110 Lahuna, O., Rastegar, M., Maiter, D., Thissen, J. P., Lemaigre, F. P. and Rousseau, G. G.
(2000) Involvement of STAT5 (signal transducer and activator of transcription 5) and
HNF-4 (hepatocyte nuclear factor 4) in the transcriptional control of the hnf6 gene by
growth hormone. Mol. Endocrinol. 14, 285–294
111 Jacquemin, P., Lemaigre, F. P. and Rousseau, G. G. (2003) The Onecut transcription
factor HNF-6 (OC-1) is required for timely specification of the pancreas and acts
upstream of Pdx-1 in the specification cascade. Dev. Biol. 258, 105–116
112 Maestro, M. A., Boj, S. F., Luco, R. F., Pierreux, C. E., Cabedo, J., Servitja, J. M.,
German, M. S., Rousseau, G. G., Lemaigre, F. P. and Ferrer, J. (2003) Hnf6 and Tcf2
(MODY5) are linked in a gene network operating in a precursor cell domain of the
embryonic pancreas. Hum. Mol. Genet. 12, 3307–3314
577
113 Jacquemin, P., Durviaux, S. M., Jensen, J., Godfraind, C., Gradwohl, G., Guillemot, F.,
Madsen, O. D., Carmeliet, P., Dewerchin, M., Collen, D. et al. (2000) Transcription factor
hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and
controls expression of the proendocrine gene ngn3. Mol. Cell. Biol. 20, 4445–4454
114 Clotman, F., Lannoy, V. J., Reber, M., Cereghini, S., Cassiman, D., Jacquemin, P.,
Roskams, T., Rousseau, G. G. and Lemaigre, F. P. (2002) The onecut transcription factor
HNF6 is required for normal development of the biliary tract. Development 129,
1819–1828
115 Odom, D. T., Zizlsperger, N., Gordon, D. B., Bell, G. W., Rinaldi, N. J., Murray, H. L.,
Volkert, T. L., Schreiber, J., Rolfe, P. A., Gifford, D. K. et al. (2004) Control of pancreas
and liver gene expression by HNF transcription factors. Science 303, 1378–1381
116 Riera, L., Manzano, A., Navarro-Sabate, A., Perales, J. C. and Bartrons, R. (2002) Insulin
induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells.
Biochim. Biophys. Acta 1589, 89–92
117 Minchenko, A., Leshchinsky, I., Opentanova, I., Sang, N., Srinivas, V., Armstead, V. and
Caro, J. (2002) Hypoxia-inducible factor-1-mediated expression of the 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3 ) gene: its possible role in the
Warburg effect. J. Biol. Chem. 277, 6183–6187
118 Barford, D., Hu, S. H. and Johnson, L. N. (1991) Structural mechanism for glycogen
phosphorylase control by phosphorylation and AMP. J. Mol. Biol. 218, 233–260
119 Hurley, J. H., Dean, A. M., Thorsness, P. E., Koshland, Jr, D. E. and Stroud, R. M. (1990)
Regulation of isocitrate dehydrogenase by phosphorylation involves no long-range
conformational change in the free enzyme. J. Biol. Chem. 265, 3599–3602
120 Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, Jr, D. E. and Stroud, R. M. (1990)
Regulation of an enzyme by phosphorylation at the active site. Science 249, 1012–1016
121 Johnson, L. N., Lowe, E. D., Noble, M. E. M. and Owen, D. J. (1998) The structural basis
for substrate recognition and control by protein kinases. FEBS Lett. 430, 1–11
122 Murray, K. J., El-Maghrabi, M. R., Kountz, P. D., Lukas, T. J., Soderling, T. R. and Pilkis,
S. J. (1984) Amino acid sequence of the phosphorylation site of rat liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J. Biol. Chem. 259,
7673–7681
123 El-Maghrabi, M. R., Claus, T. H., Pilkis, J. and Pilkis, S. J. (1982) Regulation of
6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.
Proc. Natl. Acad. Sci. U.S.A. 79, 315–319
124 Sakakibara, R., Kitajima, S. and Uyeda, K. (1984) Differences in kinetic properties of
phospho and dephospho forms of fructose-6-phosphate, 2-kinase and fructose
2,6-bisphosphatase. J. Biol. Chem. 259, 41–46
125 Van Schaftingen, E., Davies, D. R. and Hers, H. G. (1981) Inactivation of
phosphofructokinase 2 by cyclic AMP-dependent protein kinase. Biochem. Biophys.
Res. Commun. 103, 362–368
126 Van Schaftingen, E., Davies, D. R. and Hers, H. G. (1982) Fructose-2,6-bisphosphatase
from rat liver. Eur. J. Biochem. 124, 143–149
127 El-Maghrabi, M. R., Claus, T. H., Pilkis, J., Fox, E. and Pilkis, S. J. (1982) Regulation of
rat liver fructose 2,6-bisphosphatase. J. Biol. Chem. 257, 7603–7607
128 Kurland, I. J., El-Maghrabi, M. R., Correia, J. J. and Pilkis, S. J. (1992) Rat liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: properties of phospho- and
dephospho-forms and of two mutants in which Ser32 has been changed by site-directed
mutagenesis. J. Biol. Chem. 267, 4416–4423
129 Abe, Y. and Uyeda, K. (1994) Effect of adding phosphorylation sites for cAMP-dependent
protein kinase to rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Biochemistry 33, 5766–5771
130 El-Maghrabi, M. R., Pate, T. M., Murray, K. J. and Pilkis, S. J. (1984) Differential effects
of proteolysis and protein modification on the activities of 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase. J. Biol. Chem. 259, 13096–13103
131 Kurland, I., Li, L., Lange, A. J., Correia, J. J., El-Maghrabi, M. R. and Pilkis, S. J. (1993)
Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate: role of the
NH2 -terminal region. J. Biol. Chem. 268, 14056–14064
132 Lin, K., Kurland, I. J., Li, L., Lee, Y. H., Okar, D., Marecek, J. F. and Pilkis, S. J. (1994)
Evidence for NH2 - and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase. J. Biol. Chem. 269, 16953–16960
133 Zhu, Z., Ling, S., Yang, Q.-H. and Li, L. (2001) Involvement of the chicken liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444 -Arg-Glu-Arg
in modulation of the bisphosphatase activity by its kinase domain. Biochem. J. 357,
513–520
134 Kitamura, K., Kangawa, K., Matsuo, H. and Uyeda, K. (1988) Phosphorylation of
myocardial fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase by cAMPdependent protein kinase and protein kinase C: activation by phosphorylation and amino
acid sequences of the phosphorylation sites. J. Biol. Chem. 263, 16796–16801
135 Rider, M. H., Vandamme, J., Lebeau, E., Vertommen, D., Vidal, H., Rousseau, G. G.,
Vandekerckhove, J. and Hue, L. (1992) The two forms of bovine heart 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase result from alternative splicing. Biochem. J. 285,
405–411
c 2004 Biochemical Society
578
M. H. Rider and others
136 Rider, M. H., Van Damme, J., Vertommen, D., Michel, A., Vandekerckhove, J. and
Hue, L. (1992) Evidence for new phosphorylation sites for protein kinase C and cyclic
AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase. FEBS Lett. 310, 139–142
137 Deprez, J., Vertommen, D., Alessi, D. R., Hue, L. and Rider, M. H. (1997)
Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B
and other protein kinases of the insulin signalling cascades. J. Biol. Chem. 272,
17269–17275
138 Bertrand, L., Alessi, D. R., Deprez, J., Deak, M., Viaene, E., Rider, M. H. and Hue, L.
(1999) Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and
Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not
protein kinase B. J. Biol. Chem. 274, 30927–30933
139 Abe, Y., Minami, Y., Li, Y., Nguyen, C. and Uyeda, K. (1995) Expression of bovine heart
fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase and determination of the
role of the carboxyl terminus by mutagenesis. Biochemistry 34, 2553–2559
140 Kessler, R. and Eschrich, K. (1996) Ser644 is important for catalytic activity but is not
involved in cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase.
FEBS Lett. 395, 225–227
141 Dihazi, H., Kessler, R. and Eschrich, K. (2003) Glucose-induced stimulation of the
Ras-cAMP pathway in yeast leads to multiple phosphorylations and activation of
6-phosphofructo-2-kinase. Biochemistry 42, 6275–6282
142 Hardie, D. G., Carling, D. and Carlson, M. (1998) The AMP-activated/SNF1 protein
kinase subfamily: metabolic sensors of the eukaryotic cell? Annu. Rev. Biochem. 67,
821–855
143 Dihazi, H., Kessler, R. and Eschrich, K. (2001) Phosphorylation and inactivation of yeast
6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypoxic
stress. Biochemistry 40, 14669–14678
144 Macdonald, F. D. and Buchanan, B. B. (1990) The role of fructose-2,6-bisphosphate in
plant tissues. In Fructose-2,6-bisphosphate (Pilkis, S. J., ed.), pp. 193–210, CRC Press,
Boca Raton
145 Stitt, M. (1990) Fructose-2,6-bisphosphate as a regulatory molecule in plants.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 153–185
146 Furumoto, T., Teramoto, M., Inada, N., Ito, M., Nishida, I. and Watanabe, A. (2001)
Phosphorylation of a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase 2-phosphatase, is regulated physiologically and developmentally in
rosette leaves of Arabidopsis thaliana . Plant Cell Physiol. 42, 1044–1048
147 Boscá, L. and Storey, K. B. (1991) Inactivation of 6-phosphofructo-2-kinase during
anaerobiosis in the marine whelk Busycon canaliculatum . Am. J. Physiol. 260,
R1168–R1175
148 Garrison, J. C., Johnsen, D. E. and Campanile, C. P. (1984) Evidence for the role of
phosphorylase kinase, protein kinase C, and other Ca2+ -sensitive protein kinases in the
response of hepatocytes to angiotensin II and vasopressin. J. Biol. Chem. 259,
3283–3292
149 Hue, L., Van Schaftingen, E. and Blackmore, P. F. (1981) Stimulation of glycolysis and
accumulation of a stimulator of phosphofructokinase in hepatocytes incubated with
vasopressin. Biochem. J. 194, 1023–1026
150 Crepin, K. M., De Cloedt, M., Vertommen, D., Foret, D., Michel, A., Rider, M. H.,
Rousseau, G. G. and Hue, L. (1992) Molecular forms of 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase expressed in rat skeletal muscle. J. Biol. Chem. 267,
21698–21704
151 Bosca, L., Challiss, R. A. and Newsholme, E. A. (1985) The effect of fructose 2,6bisphosphate on muscle fructose-1,6-bisphosphatase activity. Biochim. Biophys. Acta
828, 151–154
152 Hue, L., Blackmore, P. F., Shikama, H., Robinson-Steiner, A. and Exton, J. H. (1982)
Regulation of fructose-2,6-bisphosphate content in rat hepatocytes, perfused hearts, and
perfused hindlimbs. J. Biol. Chem. 257, 4308–4313
153 Jones, J. P., MacLean, P. S. and Winder, W. W. (1994) Correlation between fructose
2,6-bisphosphate and lactate production in skeletal muscle. J. Appl. Physiol. 76,
2169–2176
154 Winder, W. W. and Duan, C. (1992) Control of fructose 2,6-diphosphate in muscle of
exercising fasted rats. Am. J. Physiol. 262, E919–E924
155 Krause, U. and Wegener, G. (1996) Control of glycolysis in vertebrate skeletal muscle
during exercise. Am. J. Physiol. 270, R821–R829
156 Minatogawa, Y. and Hue, L. (1984) Fructose 2,6-bisphosphate in rat skeletal muscle
during contraction. Biochem. J. 223, 73–79
157 Green, H. J., Cadefau, J. and Pette, D. (1991) Altered glucose 1,6-bisphosphate and
fructose 2,6-biphosphate levels in low-frequency stimulated rabbit fast-twitch muscle.
FEBS Lett. 282, 107–109
158 Rider, M. H. and Hue, L. (1985) Regulation of fructose 2,6-bisphosphate concentration
in white adipose tissue. Biochem. J. 225, 421–428
c 2004 Biochemical Society
159 Pessin, J. E., Thurmond, D. C., Elmendorf, J. S., Coker, K. J. and Okada, S. (1999)
Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. J. Biol. Chem. 274,
2593–2596
160 Rider, M. H. and Hue, L. (1984) Activation of rat heart phosphofructokinase-2 by insulin
in vivo . FEBS Lett. 176, 484–488
161 Larner, J., Lawrence, J. C., Walkenbach, R. J., Roach, P. J., Hazen, R. J. and Huang, L. C.
(1978) Insulin control of glycogen synthesis. Adv. Cyclic Nucleotide Res. 9, 425–439
162 Ha, J., Lee, J.-K., Kim, K.-S., Witters, L. A. and Kim, K.-H. (1996) Cloning of human
acetyl-CoA carboxylase-β and its unique features. Proc. Natl. Acad. Sci. U.S.A. 93,
11466–11470
163 Lopez-Casillas, F., Bai, D.-H., Luo, X., Kong, I.-S., Hermodson, M. A. and Kim, K.-H.
(1988) Structure of the coding sequence and primary amino acid sequence of
acetyl-coenzyme A carboxylase. Proc. Natl. Acad. Sci. U.S.A. 85, 5784–5788
164 Saddik, M., Gamble, J., Witters, L. A. and Lopaschuk, G. D. (1993) Acetyl-CoA
carboxylase regulation of fatty acid oxidation in the heart. J. Biol. Chem. 268,
25836–25845
165 Awan, M. M. and Saggerson, E. D. (1993) Malonyl-CoA metabolism in cardiac myocytes
and its relevance to the control of fatty acid oxidation. Biochem. J. 295, 61–66
166 White, M. F. (1998) The IRS-signalling system: a network of docking proteins that
mediate insulin action. Mol. Cell. Biochem. 182, 3–11
167 Lizcano, J. M. and Alessi, D. R. (2002) The insulin signalling pathway. Current Biol. 12,
R236–R238
168 Vanhaesebroeck, B. and Alessi, D. R. (2000) The PI3K–PDK1 connection: more than just
a road to PKB. Biochem. J. 346, 561–576
169 Saltiel, A. R. and Kahn, C. R. (2001) Insulin signalling and the regulation of glucose and
lipid metabolism. Nature (London) 414, 799–806
170 Lefebvre, V., Méchin, M.-C., Louckx, M. P., Rider, M. H. and Hue, L. (1996) Signaling
pathway involved in the activation of heart 6-phosphofructo-2-kinase by insulin.
J. Biol. Chem. 271, 22289–22292
171 Pozuelo Rubio, M., Peggie, M., Wong, B. H., Morrice, N. and MacKintosh, C. (2003)
14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated
cardiac fructose-2,6-bisphosphate kinase/phosphatase. EMBO J. 22, 3514–3523
172 Mora, A., Davies, A. M., Bertrand, L., Sharif, I., Budas, G. R., Jovanovic, S., Mouton, V.,
Kahn, C. R., Lucocq, J. M., Gray, G. A. et al. (2003) Deficiency of PDK1 in cardiac muscle
results in heart failure and increased sensitivity to hypoxia. EMBO J. 22, 4666–4676
173 Deprez, J., Bertrand, L., Alessi, D. R., Krause, U., Hue, L. and Rider, M. H. (2000) Partial
purification of a wortmannin-sensitive and insulin-stimulated protein kinase that
activates heart 6-phosphofructo-2-kinase. Biochem. J. 347, 305–312
174 Newton, A. C. (2001) Protein kinase C: structural and spatial regulation by
phosphorylation, cofactors, and macromolecular interactions. Chem. Rev. 101,
2353–2369
175 Narabayashi, H., Lawson, J. W. and Uyeda, K. (1985) Regulation of phosphofructokinase
in perfused rat heart: requirement for fructose 2,6-bisphosphate and a covalent
modification. J. Biol. Chem. 260, 9750–9758
176 Russell, R. R., Bergeron, R., Shulman, G. I. and Young, L. H. (1999) Translocation of
myocardial GLUT-4 and increased glucose uptake through activation of AMPK by
AICAR. Am. J. Physiol. 277, H643–H649
177 Marsin, A.-S., Bertrand, L., Rider, M. H., Deprez, J., Beauloye, C., Vincent, M. F.,
Van Den Berghe, G., Carling, D. and Hue, L. (2000) Phosphorylation and activation of
heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia.
Curr. Biol. 10, 1247–1255
178 Hue, L. (1982) Role of fructose 2,6-bisphosphate in the stimulation of glycolysis by
anoxia in isolated hepatocytes. Biochem. J. 206, 359–365
179 Depré, C., Rider, M. H., Veitch, K. and Hue, L. (1993) Role of fructose 2,6-bisphosphate
in the control of heart glycolysis. J. Biol. Chem. 268, 13274–13279
180 Beauloye, C., Marsin, A. S., Bertrand, L., Vanoverschelde, J. L., Rider, M. H. and Hue, L.
(2002) The stimulation of heart glycolysis by increased workload does not require AMPactivated protein kinase but a wortmannin-sensitive mechanism. FEBS Lett. 531,
324–328
181 Marsin, A. S., Bouzin, C., Bertrand, L. and Hue, L. (2002) The stimulation of glycolysis
by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and
inducible 6-phosphofructo-2-kinase. J. Biol. Chem. 277, 30778–30783
182 Okamura, N. and Sakakibara, R. (1998) A common phosphorylation site for cyclic
AMP-dependent protein kinase and protein kinase C in human placental
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biosci. Biotechnol. Biochem.
62, 2039–2042
183 Almeida, A., Moncada, S. and Bolanos, J. P. (2004) Nitric oxide switches on glycolysis
through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat. Cell Biol.
6, 45–51
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glycolysis
184 Loiseau, A. M., Rider, M. H., Foret, D., Rousseau, G. G. and Hue, L. (1988) Rat hepatoma
(HTC) cell 6-phosphofructo-2-kinase differs from that in liver and can be separated from
fructose 2,6-bisphosphatase. Eur. J. Biochem. 175, 27–32
185 Denis-Pouxviel, C., Gauthier, T., Daviaud, D. and Murat, J. C. (1990)
Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line:
regulation by insulin and phorbol esters. Biochem. J. 268, 465–470
186 Nissler, K., Petermann, H., Wenz, I. and Brox, D. (1995) Fructose 2,6-bisphosphate
metabolism in Ehrlich ascites tumour cells. J. Cancer Res. Clin. Oncol. 121, 739–745
187 Hirata, T., Kato, M., Okamura, N., Fukasawa, M. and Sakakibara, R. (1998) Expression of
human placental-type 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase in
various cells and cell lines. Biochem. Biophys. Res. Commun. 242, 680–684
188 Martin-Sanz, P., Cascales, M. and Bosca, L. (1992) Characterization of 6-phosphofructo2-kinase from foetal-rat liver. Biochem. J. 281, 457–463
189 Rosa, J. L., Ventura, F., Carreras, J. and Bartrons, R. (1990) Fructose 2,6-bisphosphate
and 6-phosphofructo-2-kinase during liver regeneration. Biochem. J. 270, 645–649
190 Bosca, L., Mojena, M., Diaz-Guerra, J. M. and Marquez, C. (1988) Phorbol
12,13-dibutyrate and mitogens increase fructose 2,6-bisphosphate in lymphocytes.
Comparison of lymphocyte and rat-liver 6-phosphofructo-2-kinase. Eur. J. Biochem.
175, 317–323
191 Casado, M., Bosca, L. and Martin-Sanz, P. (1996) Multiple forms of 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase are expressed in perinatal rat liver.
Am. J. Physiol. 270, E244–E250
192 Joaquin, M., Rosa, J. L., Bartrons, R. and Tauler, A. (1997) Expression of the F-type
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA during liver
regeneration. Biochim. Biophys. Acta 1334, 256–260
193 Bosca, L., Mojena, M., Ghysdael, J., Rousseau, G. G. and Hue, L. (1986) Expression of
the v-src or v-fps oncogene increases fructose 2,6-bisphosphate in chick-embryo
fibroblasts: novel mechanism for the stimulation of glycolysis by retroviruses.
Biochem. J. 236, 595–599
194 Marchand, M. J., Maisin, L., Hue, L. and Rousseau, G. G. (1992) Activation of
6-phosphofructo-2-kinase by pp60v-src is an indirect effect. Biochem. J. 285, 413–417
195 Bosca, L., Rousseau, G. G. and Hue, L. (1985) Phorbol 12-myristate 13-acetate and
insulin increase the concentration of fructose 2,6-bisphosphate and stimulate glycolysis
in chicken embryo fibroblasts. Proc. Natl. Acad Sci. U.S.A. 82, 6440–6444
196 Meacci, E., Vasta, V., Vannini, F., Farnararo, M. and Bruni, P. (1994) Bradykinin
stimulates fructose 2,6-bisphosphate metabolism in human fibroblasts.
Biochim. Biophys. Acta 1221, 233–237
579
197 Vasta, V., Bruni, P. and Farnararo, M. (1987) Mechanism of thrombin-induced rise in
platelet fructose 2,6-bisphosphate content: studies using phorbol myristate acetate,
dioctanoylglycerol and ionophore A23187. Biochem. J. 244, 547–551
198 Meacci, E., Vasta, V., Farnararo, M. and Bruni, P. (1996) Fructose 2,6-bisphosphate
metabolism during megakaryocytic differentiation of K562 and MEG-01 cells.
Mol. Cell. Biochem. 156, 125–130
199 Colomer, D., Vives Corrons, J. L. and Bartrons, R. (1991) Effect of TPA on fructose
2,6-bisphosphate levels and protein kinase C activity in B-chronic lymphocytic leukemia
(B-CLL). Biochim. Biophys. Acta 1097, 270–274
200 Cascales, M., Martin-Sanz, P. and Bosca, L. (1992) Phorbol esters, bombesin and
insulin elicit differential responses on the 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase system in primary cultures of foetal and adult rat hepatocytes.
Eur. J. Biochem. 207, 391–397
201 Kolch, W. (2000) Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK
pathway by protein interactions. Biochem. J. 351, 289–305
202 Baulida, J., Onetti, R. and Bassols, A. (1992) Effects of epidermal growth factor on
glycolysis in A431 cells. Biochem. Biophys. Res. Commun. 183, 1216–1223
203 Farnararo, M., Vasta, V., Bruni, P. and D’Alessandro, A. (1984) The effect of insulin on
Fru-2,6-P2 levels in human fibroblasts. FEBS Lett. 171, 117–120
204 Hamer, M. J. and Dickson, A. J. (1990) Control of glycolysis in cultured chick embryo
hepatocytes: fructose 2,6-bisphosphate content and phosphofructokinase-1 activity are
stimulated by insulin and epidermal growth factor. Biochem. J. 269, 685–690
205 Wu, C., Okar, D. A., Newgard, C. B. and Lange, A. J. (2001) Overexpression of
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in mouse liver lowers blood
glucose by suppressing hepatic glucose production. J. Clin. Invest. 107, 91–98
206 Baltrusch, S., Lenzen, S., Okar, D. A., Lange A. J. and Tiedge, M. (2001) Characterization
of glucokinase-binding protein epitopes by a phage-displayed peptide library:
identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel
interaction partner. J. Biol. Chem. 276, 43915–43923
207 Baltrusch, S., Okar, D. A., Lange, A. J., Lenzen, S. and Tiedge, M. (2004) Interaction of
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with glucokinase activates
glucose phosphorylation and glucose metabolism in insulin-producing cells. Diabetes
4, 1020–1029
208 Thompson, J. D., Gibson, T. J., Plewniak, J., Jeanmougin, F. and Higgins, D. G. (1997)
The ClustalX windows interface: flexible strategies for multiple sequence alignment
aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882
Received 7 May 2004; accepted 1 June 2004
Published as BJ Immediate Publication 1 June 2004, DOI 10.1042/BJ20040752
c 2004 Biochemical Society