Biochem. J. (2013) 452, 369–379 (Printed in Great Britain)
369
doi:10.1042/BJ20130118
REVIEW ARTICLE
Inositol pyrophosphates: between signalling and metabolism
Miranda S. C. WILSON, Thomas M. LIVERMORE and Adolfo SAIARDI1
Medical Research Council (MRC) Cell Biology Unit and Laboratory for Molecular Cell Biology, Department of Cell and Developmental Biology, University College London, Gower Street,
London WC1E 6BT, U.K.
The present review will explore the insights gained into
inositol pyrophosphates in the 20 years since their discovery
in 1993. These molecules are defined by the presence of
the characteristic ‘high energy’ pyrophosphate moiety and can
be found ubiquitously in eukaryotic cells. The enzymes that
synthesize them are similarly well distributed and can be found
encoded in any eukaryote genome. Rapid progress has been
made in characterizing inositol pyrophosphate metabolism and
they have been linked to a surprisingly diverse range of cellular
functions. Two decades of work is now beginning to present a
view of inositol pyrophosphates as fundamental, conserved and
highly important agents in the regulation of cellular homoeostasis.
In particular it is emerging that energy metabolism, and thus ATP
production, is closely regulated by these molecules. Much of
the early work on these molecules was performed in the yeast
Saccharomyces cerevisiae and the social amoeba Dictyostelium
INTRODUCTION
The inositol phosphate family represents one of the most
important signalling families, using myo-inositol as the basic
building block. Each of the six hydroxy groups of the inositol
ring can be phosphorylated in a combinatorial manner, generating
many signalling molecules, the best understood of which is
the Ca2 + release factor IP3 (myo-inositol 1,4,5-trisphosphate)
[1,2]. Two subfamilies of inositol phosphate exist: the lipidbound, commonly called PIs (phosphatidylinositols), of which
seven species have been identified; and the cytosolic inositol
phosphates, extracted from cells using water-based acid solutions
and thus referred to as ‘soluble’. The traditional method to
resolve the soluble inositol phosphates relies on their differential
negative charge. Owing to the different number and position
of the phosphate groups, different members of the family
elute at different times during SAX (strong anion exchange)
chromatography [3]. Peaks eluting after the fully phosphorylated
ring of IP6 (inositol hexakisphosphate; also known as phytic acid)
were first noted in the early 1990s [4–7] and were suggestive of
a molecule more polar still than IP6. The discovery of inositol
discoideum, but the development of mouse knockouts for IP6K1
and IP6K2 [IP6K is IP6 (inositol hexakisphosphate) kinase] in the
last 5 years has provided very welcome tools to better understand
the physiological roles of inositol pyrophosphates. Another recent
innovation has been the use of gel electrophoresis to detect
and purify inositol pyrophosphates. Despite the advances that
have been made, many aspects of inositol pyrophosphate biology
remain far from clear. By evaluating the literature, the present
review hopes to promote further research in this absorbing area
of biology.
Key words: bisdiphosphoinositol tetrakisphosphate (IP8 ),
5-diphosphoinositol pentakisphosphate (IP7 ), inositol hexakisphosphate (IP6 ) kinase (IP6K), inositol phosphate, metabolism,
polymer inorganic polyphosphate (polyP), PP-IP5 kinase
(PPIP5K).
pyrophosphates, however, is generally accredited to two papers,
from two research groups, both published in the Journal of
Biological Chemistry in early 1993 [8,9].
Phytic
acid
is
the
precursor
of
the
best
characterized inositol pyrophosphates, IP7 (or PP-IP5 ; 5diphosphoinositol pentakisphosphate) and IP8 [or (PP)2 IP4 ;
bisdiphosphoinositol tetrakisphosphate], that possess, as the
name suggests, seven or eight phosphate groups attached to
the six-carbon inositol ring, thus possessing one and two
pyrophosphate moieties respectively (Figure 1). Although the
pyro- nomenclature is now commonly used, it is important
to mention that the correct IUPAC definition for this moiety
is diphospho-, thus the correct chemical name of IP7 is
diphosphoinositol pentakisphosphate (or PP-IP5 ). The past
20 years have seen this class of molecules as well as related
enzymes and gene names referred to in several ways. Table 1
displays these different nomenclatures, indicating the scientific
name and the common name used in the present review.
Inositol pyrophosphates have the distinctive property of
containing high-energy phosphate bonds. The free energy
of hydrolysis of the pyrophosphate moiety is similar to that of
Abbreviations used: AEC, adenylate energy charge; AMPK, AMP-activated protein kinase; AP3, adaptor protein 3; AP3B1, β-1 subunit of
the AP3 complex; ATM, ataxia telangiectasia mutated; ATR, ATM- and Rad3-related; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; Crac,
cytosolic regulator of adenylate cyclase; DIPP, diphosphoinositol-phosphate phosphohydrolase; DNA-PKcs, DNA-dependent protein kinase, catalytic
subunit; HEK, human embryonic kidney; HR, homologous recombination; IFN-β, interferon-β; IP3 , myo -inositol 1,4,5-trisphosphate; IP4 , inositol
tetrakisphosphate; IP5 , inositol pentakisphosphate; IP6 /PP-IP4 , inositol hexakisphosphate; IP7 /PP-IP5 , 5-diphosphoinositol pentakisphosphate; IP8 /(PP)2 IP4 ,
bisdiphosphoinositol tetrakisphosphate; IP3K, IP3 3-kinase; IP6K, IP6 kinase; IPMK, inositol phosphate multikinase; IPPK/IP5 -2K, IP5 -2 kinase;
MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; Nudix, nucleoside diphosphate
linked moiety X; PDK1, 3-phosphoinositide-dependent protein kinase 1; PH, pleckstrin homology; PI, phosphatidylinositol; PI3K, phosphoinositide
3-kinase; PIKK, PI3K-related; PIP2 , phosphatidylinositol 4,5-bisphosphate; PIP3 , phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; polyP,
inorganic polyphosphate; PPIP5K, PP-IP5 kinase; TOR, target of rapamycin.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
370
M. S. C. Wilson, T. M. Livermore and A. Saiardi
isomers are considered. More than 30 different soluble inositol
phosphates have been identified in eukaryotic cells [11].
In the yeast experimental model Saccharomyces cerevisiae, the
synthesis of the soluble pool of inositol phosphates begins with
the well-studied second messenger IP3 (Figure 1). This molecule
is the cleavage product of the lipid PIP2 (phosphatidylinositol
4,5-bisphosphate) by phospholipase C [12,13]. Once liberated
from its DAG (diacylglycerol) tail the soluble molecule can
be dephosphorylated to myo-inositol, or it can be further
phosphorylated to the fully phosphorylated IP6 ring and beyond,
to form the inositol pyrophosphates IP7 and IP8 (Figure 1)
[8,9]. In the model amoeba Dictyostelium discoideum, the
synthesis of the higher phosphorylated forms of inositol does not
require phospholipase C activity, and instead occurs by a
direct sequential phosphorylation route from myo-inositol to IP6
(Figure 1) [9,15].
Kinases
Figure 1
Linear pathway of inositol pyrophosphates synthesis
This pathway depicts the possible routes of inositol pyrophosphate synthesis in cells, starting
from myo -inositol. S. cerevisiae exclusively uses the lipid route to synthesize inositol phosphates
[119], whereas D. discoideum utilizes the cytosolic route to synthesize IP6 [15], the enzymology
of which is not fully elucidated. The interested reader is invited to read the reviews [11,120,121]
which describe the synthesis of IP6 in more detail. This Figure highlights the metabolic steps
most relevant in vivo , supported by yeast genetic studies [25,26,40,53,119]. There are multiple
PP-IP4 isoforms synthesized by IP6K in vitro [(1/3)PP-IP4 and 5PP-IP4 are shown here] [19].
The broken line represents the axis of symmetry through myo -inositol between carbons 2 and
5, making positions 1,3 and 5,6 enantiomeric. P indicates a single phosphate (PO3 2 − ) group;
R and R represent fatty acid chains; broken arrows represent multiple steps of modification that
are not shown in detail; and kinases catalysing each step are indicated in red (mammalian) and
blue (S. cerevisiae ).
the high-energy bond found in ATP [9,10]. Like ATP, IP7
and IP8 are found in all eukaryotic cells analysed so far,
ranging from fungi to humans. Inositol pyrophosphates control
disparate cell biological processes, from regulating telomere
length to controlling vesicular trafficking, from insulin secretion
to chemotaxy. However, an emerging hypothesis places the
inositol pyrophosphates as conserved and fundamental regulators
of cellular energy metabolism, enabling their many and varied
downstream effects. This new vision will be the driving theme of
the present review.
STRUCTURE AND SYNTHESIS
The simple myo-inositol ring forms the basis of the diverse
family of inositol phosphates. Existing in the chair formation,
this six-carbon sugar molecule has one axis of symmetry, through
its 2 and 5 positions (Figure 1). This stereochemistry arises
due to the axial nature of the hydroxy group on position 2,
whereas the other five hydroxy groups are in the equatorial
plane. An important consequence of this is the expansion of the
range of phosphorylated states that can result from this building
block. With each carbon representing a distinct stereochemical
environment, it has been calculated that there are 63 possible
varieties of inositol phosphate, even before the pyrophosphate
c The Authors Journal compilation c 2013 Biochemical Society
Inositol pyrophosphates are largely synthesized from IP6 ,
generating IP7 and IP8 . IP6 , the most abundant of the inositol
phosphates, can reach concentrations of 10–60 μM in mammalian
cells and 0.7 mM in D. discoideum [16], whereas IP7 can reach
concentrations of 0.5–1.3 μM in mammalian cells [17,18] and
0.3–0.5 mM in the amoeba [17]. Two classes of enzymes are
responsible for the synthesis of pyrophosphates: the IP6Ks
(IP6 kinases) and the PPIP5Ks (PP-IP5 kinases), Kcs1 and
Vip1 respectively in S. cerevisiae. These two distinct classes
of enzyme exhibit catalytic activity against different positions
on the ring, with IP6Ks placing a phosphate group at the
β position of C-5 on the fully phosphorylated IP6 ring [19],
whereas the PPIP5Ks phosphorylate position 1 [20,21] (J.D.
York, personal communication) (Figure 1). The isomeric form
of IP8 present in mammalian cells has, therefore, been identified
as (1,5)PP-IP4 [18] (Figure 1). Interestingly, despite similar
enzymology, D. discoideum instead contains the (5,6)PP-IP4
isomer of IP8 [10,22], possibly reflecting different metabolism.
Although in vitro PPIP5Ks do not act on the principal IP5
(inositol pentakisphosphate) isomer I(1,3,4,5,6)P5 [23,24], the
IP6K enzymes are able to phosphorylate it, mainly, but not
exclusively, at the enantiomeric positions 1,3, generating (1,3)PPIP4 . This can then be further phosphorylated by IP6K or PPIP5K to
(PP)2 -IP3 (bis-disphosphoinositol trisphosphate) [25]. Yeast Kcs1
has been shown in vivo to generate inositol pyrophosphate even
using IP3 and IP4 (inositol tetrakisphosphate) [26]. It is worthy of
mention that in vitro the IP6Ks are also able to form a triphosphate
PPP-IP5 form of ‘IP8’ [19]. Therefore taking into account the
diverse substrates and the formation of the pyrophosphate moiety
at different positions of the inositol ring, inositol pyrophosphates
have the potential to become a very large family of molecules.
The IP6Ks belong to the same family of inositol phosphate
kinases as the IP3K (IP3 3-kinase) and the IPMK (inositol
phosphate multikinase). This family is characterized by the
presence of the conserved PxxxDxKxG motif in the inositolbinding region (Pfam family PF03770) [27,28]. Phylogenetic
analysis indicates that in fact IP6Ks are likely to be the most
ancient members of this family [29]. The IP6Ks were first
characterized and cloned in the Snyder laboratory shortly after
the identification of IP7 [27,30]. Three mammalian genes, IP6K1,
IP6K2 and IP6K3, were identified [31], one of which, IP6K2,
had previously been identified as PiUS (Pi uptake stimulator)
[27,32,33]. In vitro all three mammalian IP6Ks, as well as the
yeast Kcs1, are capable of phosphorylating IP6 to IP7, as well
as IP5 to PP-IP4 [31,34,35]. The physiological relevance of these
derivatives is much debated and is likely to be highly contingent
Inositol pyrophosphates
Table 1
371
Inositol pyrophosphate nomenclature
(a) Summary of the nomenclature of the inositol pyrophosphates, with the names used in the present review in the left-hand column, the correct IUPAC nomenclature, the alternative names sporadically
present in the literature and the identified biological isomers. (b) Details of the mammalian inositol pyrophosphate synthesis genes, including the chromosomal localization of the human isoforms,
as well as previous names and the corresponding S. cerevisiae gene name.
(a)
Common name
IUPAC nomenclature
Alternative name
Isomeric forms
IP6
IP7 /PP-IP5
Inositol hexakisphosphate (InsP6 )
Diphosphoinositol pentakisphosphate (PP-InsP5 )
Phytic acid
InsP7
IP8 /(PP)2 -IP4
Bisdiphosphoinositol tetrakisphosphate [(PP)2 -InsP4 ]
InsP8
IP5
PP-IP4
Inositol pentakisphosphate [Ins(1,3,4,5,6)P5 ]
Diphosphoinositol tetrakisphosphate
InsP5
PP-InsP4
(PP)2 -IP3
Bisdiphosphoinositol trisphosphate
(PP)2 -InsP3
Enzymes
Names and human gene chromosome localization
Other names
Yeast gene
IP6K
Inositol hexakisphosphate kinase
IP6K1 (3p21.31)
IP6K2 (3p21.31)
IP6K3 (6p21.31)
Diphosphoinositol pentakisphosphate kinase
PPIP5K1 (15q15.3)
PPIP5K2 (5q21.1)
KIAA0263; IHPK1 PiUS; IHPK2 IHPK3
KCS1
hsVIP1; HISPPD2A; IPS1 hsVIP2; HISPPD1; IP7K2
VIP1
1PP-IP5
5PP-IP5
1,5PP-IP4 (mammal)
5,6PP-IP4 (D. discoideum )
(1/3)PP-IP4
5PP-IP4
(b)
PPIP5K
Figure 2
Proposed mechanisms of action for inositol pyrophosphates
The allosteric mechanism of action (top), i.e. the binding of an inositol pyrophosphate to a protein
effector, has been suggested for the S. cerevisiae protein Pho81 [67] and the PH domains of
the D. discoideum protein Crac [59] and the mammalian Akt [57]. However, this simple
binding mechanism does not make use of the pyrophosphate bond. One can hypothesize that
a protein-binding partner may cycle through conformational states by binding and hydrolysis
of an inositol pyrophosphate, similar to the regulation of GTPases. Pyrophosphorylation of
proteins (bottom) instead takes advantage of the ability of inositol pyrophosphates to participate
in phosphotransfer reactions. A pre-phosphorylated serine residue in a negatively charged local
environment (shown in red) can become the recipient of the β-phosphate of the pyrophosphate
moiety, in the presence of divalent cations [72,73].
both on the relative concentrations of IP5 and IP6 in a given cell as
well as the substrate specificity of the kinase involved. In yeast the
presence of PP-IP4 , for instance, is much increased in the ipk1
[IP5 -2K (IP5 -2 kinase); IPPK in mammals] strain that is unable to
synthesize IP6 (Figure 1) and thus has elevated levels of IP5 . It is
also clear that the relative affinity for IP6 over IP5 of a given IP6K
varies between organism and isoform; human IP6K1 for instance
exhibits a 5-fold difference in K m between IP6 and IP5 , whereas
IP6K2 has a 20-fold higher affinity for IP6 over IP5 [27].
In 2007 the York laboratory identified the second class of
pyrophosphate-producing kinases in the form of the yeast enzyme
Vip1 [36]. Two mammalian homologues, PPIP5K1 and PPIP5K2,
were also identified [23,37]. These reasonably large proteins,
approximately 150 kDa in size, do not belong to the inositol
phosphate kinase family. In addition to the kinase domain, they
also possess a histidine acid phosphatase-like domain in the
C-terminal portion of the protein. It has been postulated
that the phosphatase domain is catalytically inactive, and is
responsible for the allosteric regulation of PPIP5K by IP6 [38].
However, truncated PPIP5K constructs lacking the phosphatase
domain show increased activity when overexpressed [36],
indicating that this domain is active. It may antagonize the
kinase domain, specifically dephosphorylating 1PP-IP5 , the IP7
isomer produced by the kinase domain (J.D. York, personal
communication). Although in vitro PPIP5K can promptly
phosphorylate IP6 to 1PP-IP5 [39], in vivo this activity is masked
by phosphatase activity and cannot be easily detected, suggesting
that the major physiological target of the PPIP5Ks is IP7 , as also
suggested by the kinetic parameters [23]. This is further supported
by the rise in levels of IP7 in vip1 mutants, whereas levels of IP6
remain unchanged [40].
Phosphatases
In kcs1 yeast, the inability to detect 1PP-IP5 in vivo could
be the result of multiple activities, i.e. by the intrinsic acidic
phosphatase domain of PPIP5K, and by the action of the DIPP
(diphosphoinositol-phosphate phosphohydrolase) proteins [41].
DIPPs prefer to hydrolyse at position 1; in fact (1,5)PP2 IP4 is hydrolysed in a specific order, first position 1 and
then position 5 [42]. Four mammalian enzymes, DIPP1, 2, 3
and 4, have been identified, whereas only one DIPP protein,
Ddp1, exists in S. cerevisiae [43,44]. These proteins belong
to the large family of Nudix (nucleoside diphosphate linked
moiety X) hydrolases, which contain a 23-amino-acid catalytic
c The Authors Journal compilation c 2013 Biochemical Society
372
M. S. C. Wilson, T. M. Livermore and A. Saiardi
domain named the MutT motif or nudix box [45]. Nudix
hydrolases catalyse the cleavage of the phosphoanhydride bond in
substrates that are usually nucleoside diphosphates linked to other
molecules generally through phosphodiester bonds. Therefore
DIPPs are very promiscuous and are able to degrade inositol
pyrophosphates as well as nucleotide analogues such as Ap6A
(diadenosine hexaphosphate) [42,46]. More recently, it has been
shown that DIPPs also degrade the ubiquitous polyP (inorganic
polyphosphate) [25]. Thus the enzymes that degrade inositol
pyrophosphate [41] are also capable of acting on polyP [47–
49], a cellular phosphate reservoir important for the regulation of
energetic metabolism, as discussed below. This ability to act on
polyP is also shared by the S. cerevisiae homologue Ddp1 [25].
REGULATION OF INOSITOL PYROPHOSPHATE LEVELS
Previous reviews have emphasized the lack of evidence suggesting
that inositol pyrophosphates rapidly and dynamically respond
to a particular stimulus [50,51]. It is unlikely that inositol
pyrophosphates participate in a classical receptor-mediated
signalling event, exemplified by phospholipase C activation that
leads to the rapid hydrolysis (in seconds) of PIP2 , and a dramatic
increase in IP3 [12]. However, many experiments have been
identified describing modulation of the cellular levels of inositol
pyrophosphates [40,50–52]. Together the evidence points to a link
between inositol pyrophosphates and the cellular metabolic status.
Shortly after the discovery of inositol pyrophosphates it became
apparent that, although the cellular concentration remains largely
constant in most cells, in mammalian cells at least the turnover of
IP7 and IP8 is in fact very rapid [8,52]. The Shears laboratory, using
the phosphatase inhibitor fluoride as a metabolic trap, showed that
in a variety of mammalian cells 50 % of the IP6 pool, as well as
20 % of the IP5 pool, were converted into pyrophosphates each
hour [8]. Furthermore the IP7 pool in primary hepatocytes was
demonstrated to turn over ten times every 40 min, compared with
just 10 % of the IP6 pool in the same time [52]. Although these
observations have led to suggestions that inositol pyrophosphates
have a dynamic role with a potential ‘molecular switch’ activity
[53], the physiological significance remains unknown, mainly
because fluoride is a commonly used protein phosphatase inhibitor
and thus has multiple effects on cell signalling. It has been
assumed that the stabilizing effect of fluoride reflects its ability to
inhibit the activity of DIPP phosphatases [41]. However, fluoride
does not elevate the inositol pyrophosphate concentration in yeast,
where Ddp1 is also sensitive to fluoride inhibition [24]. Similarly,
fluoride does not alter the inositol pyrophosphate level in
D. discoideum or plant cells (A. Saiardi, unpublished work),
thus the effect of fluoride seems to be specific to mammalian
cells, where the mechanism is unknown. Aside from fluoride,
a few other treatments have been shown to regulate inositol
pyrophosphates in mammalian cells. Induction of apoptosis in
ovarian cancer cells by 8 h of cisplatin or staurosporine treatment
led to a 4-fold increase in IP7 levels, and a slight increase in IP8
[54]. Inositol pyrophosphate levels also change during the cell
cycle. In rat mammary tumour cells, the level of IP7 was twice
as high in G1 -phase as in the other phases of the cell cycle [55].
Variation in the IP7 level through the cell cycle was also recently
seen in yeast, although here IP7 peaked in S-phase rather than
G1 -phase [56].
In MEFs (mouse embryonic fibroblasts), overnight serumstarvation depleted IP7 levels, which could be restored with 1 h of
IGF-1 (insulin-like growth factor 1) treatment [57]. Interestingly,
starvation also induces the most dramatic ‘physiological’ change
in inositol pyrophosphate levels described so far. The social
c The Authors Journal compilation c 2013 Biochemical Society
amoeba Dictyostelium is single-celled while food is available;
starvation induces a cAMP-mediated chemotaxic aggregation into
a multicellular ‘slug’, followed by further differentiation into a
fruiting body. Starvation has been reported to increase IP7 and
IP8 levels up to 25-fold within hours of nutrient removal [58,59].
A newer methodology for investigating endogenous unlabelled
inositol phosphate levels in the amoeba also found an increase
in inositol pyrophosphates during development, but to a much
lower level (A. Saiardi and T.M. Livermore, unpublished work).
Treatment with cAMP directly has been shown to rapidly elevate
IP7 and IP8 levels in Dictyostelium within 60 s, although the
cells had been pre-starved for 5 h and treated with caffeine, an
adenylate cyclase and PIKK [PI3K (phosphoinositide 3-kinase)related] family inhibitor that was used to reduce endogenous
cAMP synthesis [59].
Nutrient starvation also regulates inositol pyrophosphate levels
in yeast. It has been reported that a decrease in extracellular
phosphate results in an increase in IP7 in S. cerevisiae [60].
However, the observation of the opposite result [24], i.e. a decrease
in IP7 levels after phosphate starvation, is easier to explain as this
treatment dramatically decreases ATP levels [61] and therefore IP7
synthesis. Restoring the phosphate concentration in the medium
has an immediate effect in restoring ATP synthesis and leads to a
rapid increase in inositol pyrophosphate levels [25].
Inositol pyrophosphates as metabolic regulators
Rather than demonstrating a role for inositol pyrophosphates
as classical second messengers, these data showing alterations
in inositol pyrophosphate levels are more coherent with a
view of pyrophosphates as metabolic messengers or energetic
sensors. In light of this new definition, the apparent induction
of IP7 in response to insulin signalling can be explained as
the levels of IP7 returning to baseline after serum-deprivation
and therefore low metabolic rate. Similarly, the changes seen
throughout the starvation-induced development of Dictyostelium
may reflect adaptations to the metabolic state of the starved
cells. Hyperosmotic and thermal stress were shown to specifically
induce IP8 in mammalian cells, but not in S. cerevisiae [62,63].
This was initially thought to be regulated by the MAPKs (mitogenactivated protein kinases), but further investigation found that the
MAPK inhibitors used were actually affecting the energy charge
of the cell, and it was this off-target effect that altered the IP8
level [64]. Pharmacological treatment with AICAR (5-amino4-imidazolecarboxamide riboside), which is processed into an
AMP mimic, caused a reduction in IP8 but not IP7 in mouse
cells. The increase was independent of AMPK (AMP-activated
protein kinase), again suggesting an independent role for inositol
pyrophosphates in energy sensing. In mammalian cells, treatment
with the Ca2 + regulator thapsigargin caused a slight decrease in
IP7 levels [52]. This is now thought to be a consequence of the
off-target reduction in ATP concentration subsequently reported
for thapsigargin treatment [51].
Support for the concept of inositol pyrophosphates, and
particularly the products of IP6K, as energy sensors can be
found in the biochemical properties of the inositol phosphate
kinases. The usual K m for ATP of the inositol phosphate kinases
IP3K, IMPK, IPPK and PPIP5K are all in the micromolar range,
approximately 20–100 μM [65]. Conversely, the IP6Ks have a
K m for ATP between 1.0 and 1.4 mM [27,30,34], similar to the
intracellular ATP concentration. This makes IP6K activity and
therefore IP7 synthesis extremely sensitive to fluctuations in the
intracellular ATP level. The recent review by Wundenberg and
Mayr suggests an elegant ‘energostatic’ model that describes how
Inositol pyrophosphates
metabolic regulation involving inositol pyrophosphates may work
[65].
MECHANISM OF ACTION
To transduce a signal, the mechanism of action used by many
members of the inositol phosphate family of molecules is via
binding to a particular receptor, such as the IP3 receptor for IP3
[2], or binding to proteins containing specific domains such as
PH (pleckstrin homology), PX (phagocyte oxidase homology)
or FYVE (for Fab1, YOTB, Vac1 and EEA1) domains [66].
Consequently inositol pyrophosphates may also signal through
allosteric interactions with proteins. However, the unique presence
of a highly energetic pyrophosphate bond has also opened the
possibility of phosphotransfer reactions, covalently modifying
the protein targets. The literature contains evidence for both
mechanisms of action, which are not mutually exclusive and may
coexist in cells (Figure 2).
The ability of inositol pyrophosphates to allosterically regulate
a binding partner has been proposed for PH domain-containing
proteins, such as the mammalian growth factor/insulin signalling
kinase Akt/PKB (protein kinase B), and Pho81, a yeast CKI
[CDK (cyclin-dependent kinase) inhibitor] that regulates the
phosphate response. In S. cerevisiae, the cyclin–CDK–CKI
complex Pho80–Pho85–Pho81 is involved in sensing phosphate
availability. The Vip1-generated IP7 , 1PP-IP5 , was found to
specifically bind to and inhibit this complex, whereas IP6 or
5PP-IP5 , the dominant IP7 species in most cells, had no effect
[67]. No mammalian homologues for these Pho proteins have
been discovered, so this may be a strictly S. cerevisiae-specific
regulatory mechanism, which is not conserved even in the fission
yeast Schizosaccharomyces pombe [68].
PH domain binding
The majority of work in this area has been to determine whether
inositol pyrophosphates can bind to PH domain-containing
proteins whose main ligand is the phosphoinositide lipid PIP3
(phosphatidylinositol 3,4,5-trisphosphate). In vitro IP7 , and to
a lesser extent its precursor IP6 , were able to compete with
PIP3 or labelled IP4 (the PIP3 head group) for binding to
the recombinant PH domains of several proteins, including the
D. discoideum chemotaxis protein Crac (cytosolic regulator
of adenylate cyclase) and mammalian Akt [59]. Because the
concentration of inositol pyrophosphates is unusually high in
Dictyostelium, it was suggested that the physiological regulation
of PH domain proteins by inositol pyrophosphates would be
restricted to this amoeba. However, it has previously been reported
that mammalian PDK1 (3-phosphoinositide-dependent protein
kinase 1)-mediated phosphorylation of Akt is strongly inhibited
by IP7 with an IC50 of 1 μM in the presence of PIP3 , whereas
the IC50 of IP7 inhibition decreases to an astonishing 20 nM in the
absence of PIP3 [57]. The authors propose that the direct binding
of IP7 to the PH domain of Akt is responsible for this inhibition
[57]; however, appropriate binding assays were not reported.
Because the cellular IP7 concentration is in the range 0.5–
1.3 μM, cytosolic Akt must then be constitutively bound and
inhibited by IP7 . If this is true, Akt needs to release IP7 before its
activation by PIP3 –PDK1 can take place at the plasma membrane.
Detailed inositol pyrophosphate binding studies were
performed with PDK1, which possesses an atypical PH domain
containing a large binding pocket. PDK1 was found to interact
with IP7 with a considerably lower affinity than that of IP6 .
Owing to the much higher abundance of IP6 compared with IP7 in
373
mammalian cells, it is unlikely that IP7 is a physiological ligand for
PDK1 [69]. The affinity of PDK1 for IP6 may function to retain a
pool of this protein in the cytosol. A subsequent study by the same
laboratory failed to identify any significant binding between the
PH domain of Akt and IP7 . Using a FRET (fluorescence resonance
energy transfer)-based assay the authors concluded that IP6 and
IP7 are not able to displace PIP3 from Akt pre-incubated with
the lipid [70]. Despite much work in this area, this aspect of
inositol pyrophosphate activity remains controversial, with the
main conceptual problems based on structural considerations.
Structural analysis of the PH domain from β-spectrin bound
to IP3 revealed that binding specificity is determined by the
phosphate groups in positions 4 and 5 [71]. It is difficult to
envision how 5PP-IP5 , with a pyrophosphate moiety in position 5,
would fit into a binding pocket designed for only one phosphate
at that position. These studies do not paint a clear picture of
inositol pyrophosphates acting primarily as ligands of PH domaincontaining proteins. Further work will be required to better define,
structurally and functionally, the binding pocket recognizing
inositol pyrophosphates. It is not clear if the many diverse inositol
pyrophosphate species would require specific receptors, and if not,
without a specific binding site for each pyrophosphate moiety, how
specificity between the pyrophosphate and the precursor would
be obtained.
Protein pyrophosphorylation
The published allosteric mechanisms so far also show a lack
of target conservation between humans, yeast and amoeba, at
odds with the near ubiquitous evolutionary appearance of IP7 .
It is possible that the primary mechanism of action of inositol
pyrophosphates is not binding, but instead relies on the ability
of the energetic pyrophosphate group to donate its β-phosphate
to acceptor molecules. It has long been suggested that inositol
pyrophosphates may act as phosphorylating agents, due to the high
free energy of hydrolysis of their pyrophosphate bond(s), which
in 1PP-IP5 is comparable with that of ADP [9,10]. This was first
tested in vitro using 32 P-labelled IP7 . When added to eukaryotic
cell extracts from yeast, mouse and flies, labelled proteins were
obtained [72]. This was enhanced with fluoride but reduced by
treatment with λ-phosphatase. The authors [72] focussed on one
candidate protein, Nsr1, a nucleolar yeast protein, although the
mammalian nucleolar proteins Nopp140 and TCOF1 were also
able to be pyrophosphorylated by labelled IP7 . Surprisingly, the
transfer of the 32 P-labelled β-phosphate to Nsr1 was found to
be non-enzymatic and did not require any known yeast kinases.
However, this modification is not untargeted, and is thought
to require serine residues within an acidic region of primary
structure. Importantly, endogenous labelling of tagged Nsr1 in
yeast given labelled Pi was much reduced in the kcs1 mutant,
suggesting that IP7 does phosphorylate Nsr1 in vivo.
A follow-up study demonstrated that IP7 was in fact
acting on pre-phosphorylated serine residues, resulting in
pyrophosphorylation [73]. Other inositol pyrophosphates, 1PPIP5 and IP8 , were found to have similar pyrophosphorylating
ability as IP7 . The free energy of hydrolysis of the pyrophosphate
bond in IP8 is greater than that of IP7 and so this molecule may
well be more efficient at modifying proteins, although in vivo IP7
is more abundant. The serine pyrophosphorylation was found
to be more acid-labile than traditional serine phosphorylation
by ATP, but more resistant to phosphatases. In fact, no protein
pyrophosphatase has yet been identified. The physiological effects
of pyrophosphorylation by IP7 and other inositol pyrophosphates
are not fully understood, although the occurrence of this
c The Authors Journal compilation c 2013 Biochemical Society
374
Figure 3
M. S. C. Wilson, T. M. Livermore and A. Saiardi
Inositol pyrophosphates: the role in metabolism
Primary metabolic influences exerted by inositol pyrophosphates (shown as IP7 ). The Figure
shows three, probably connected, features that link inositol pyrophosphates to metabolism at the
molecular (phosphate homoeostasis), cellular (energetic) and organismal (insulin signalling)
levels.
modification is conserved and has, so far, been demonstrated in
both mammalian [74,75] and zebrafish (A. Saiardi, unpublished
work) cells. Two studies have been published investigating how
pyrophosphorylation may modulate protein activity in yeast and
human cells [74,75]. Both demonstrated that pyrophosphorylation
mediates and inhibits protein–protein interactions. In these
investigations, pyrophosphorylation resulted in regulation of yeast
glycolysis, and the release of HIV-Gag virus-like particles from
HeLa cells.
Like the allosteric mechanism of action, the protein
pyrophosphorylation mechanism also lacks the support of
strong experimental evidence, proving that this novel posttranslational protein modification occurs in vivo. The strongest
evidence indicating that inositol pyrophosphates covalently
modify proteins in vivo relies on the fact that many substrates
of in vitro phosphorylation by IP7 show an upward gelmobility shift in extracts from wild-type compared with kcs1
yeast [74]. However, no direct biophysical evidence of this
modification exists in the literature, thus further work is required
to elucidate the exact nature of the covalent modification driven
by pyrophosphates in vivo.
FUNCTION
Inositol pyrophosphates have been implicated in a variety of
diverse activities, from apoptosis and telomere maintenance
to trafficking and embryonic development [53,54,76,77]. This
diversity and wide range of activities underlines the biological
importance of this class of molecules; until recently, it
was unclear whether the inositol pyrophosphates regulated
each reported cellular activity independently, or if they were
controlling some central basic function that sits upstream of
the regulated phenotypes. First by theoretical considerations
[29], then subsequently on the basis of published results, it
has been proposed that inositol pyrophosphates have a role as
‘metabolic messengers’ [51] or ‘regulators of cell homoeostasis’
[65]. The study by Szijgyarto et al. [75] indeed suggests that
inositol pyrophosphates are master regulators of cellular energetic
metabolism. In this section, we will summarize the literature so
far regarding the function of inositol pyrophosphates, with a focus
on this concept of metabolic regulation (Figure 3).
As described above, the synthesis of inositol pyrophosphates
is tied to ATP levels due to the high K m for ATP of the
IP6K enzymes. Surprisingly, cells with no or a decreased
c The Authors Journal compilation c 2013 Biochemical Society
amount of inositol pyrophosphates, such as kcs1 yeast and
IP6K1 − / − MEFs, were found to have a greatly increased
cellular ATP concentration, whereas ADP and AMP levels were
slightly reduced, leading to an overall increase in the adenine
nucleotide pools (adenylate pool) and more importantly to an
overall increase in AEC {adenylate energy charge; calculated by
([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP])} [75]. The AEC
corresponds to the saturation of phosphodiesteric bonds of the
adenylate pool, and thus represents the amount of metabolically
available energy stored in the adenine nucleotide pool [78].
Conversely, increasing the amount of IP7 in wild-type yeast
by expressing mammalian IP6K1 caused a decrease in ATP
concentration. The yeast system was studied in more detail and it
was discovered that protein pyrophosphorylation was influencing
the interactions of the essential yeast glycolytic transcription
factors Gcr1, Gcr2 and Rap1 [79,80]. Pyrophosphorylation of
Gcr1 reduced its ability to bind Gcr2, thereby inhibiting the
transcription of genes encoding glycolytic enzymes. Accordingly,
the kcs1 yeast consumed glucose more quickly than wild-type
yeast [75].
Surprisingly, given the increase in cellular ATP concentration,
the mitochondria were found to be defective in the kcs1
yeast. Yeast usually generate energy and intermediates through
fermentation, but can be induced to perform mitochondrial
metabolism/oxidative phosphorylation by providing only a nonfermentable carbon source such as glycerol; the kcs1 yeast
were unable to grow on this medium. That yeast preferentially
perform fermentation marks them out compared with higher
eukaryotes, and the Gcr1 and Gcr2 proteins are not found in
mammals. However, the increased ATP level and reduced oxygen
consumption of IP6K1 − / − MEFs compared with wild-type
MEFs demonstrates that the inositol pyrophosphate regulation
of fundamental cellular metabolism is evolutionarily conserved.
ATP availability, along with NAD/NADH levels, which were also
altered, underpins virtually all other cellular activity.
Inositol pyrophosphates also regulate D. discoideum
chemotaxis [59]. In this amoeba starvation and subsequent cAMP
signalling and chemotaxis causes development into a multicellular
fruiting body. The D. discoideum ip6k knockout was more
sensitive to cAMP and aggregated more quickly than wild-type
cells in response to starvation [59]. It was suggested that this
phenotype reflected competition in the wild-type cells between
IP7 and PIP3 for binding to the PH domain of Crac, a protein
linked to cAMP-dependent chemotaxis. As discussed above, the
evidence describing the ability of inositol pyrophosphates to
compete with lipid inositols for PH domain binding is conflicting.
In the case of Dictyostelium aggregation, PIP3 is unlikely to be
the chief regulatory molecule: deletion of all five PI3Ks capable
of generating PIP3 did not prevent chemotaxis to cAMP [81].
However, if IP7 affects energetic metabolism and the adenine
nucleotide level in this system as in yeast, the high level of ATP
may lead to an increased cAMP concentration and subsequently
the faster chemotaxic response observed in the D. discoideum
ip6k-null strain.
IP6K − / − mouse characterization
Further work has indicated a role for inositol pyrophosphates
in metabolic regulation not only at the cellular, but also at the
organismal level. Overexpression of IP6K1 in pancreatic β-cells
or direct addition of IP7 resulted in an increase in insulin vesicle
membrane fusion, i.e. exocytosis [82], whereas knockdown
of IP6K1 by RNAi (RNA interference) reduced exocytosis
in these cells. This finding was supported by the IP6K1 − / −
Inositol pyrophosphates
mouse phenotype: it has reduced plasma insulin compared with
the wild-type mouse [83]. However, the mutant mouse also
displays insulin hypersensitivity, responding more strongly to
lower concentrations of insulin [57]. The IP6K1 − / − mouse is
slightly smaller than the wild-type mouse on a normal diet, owing
to a reduction in fatty tissue levels. Even when the mice were fed
a high-fat diet, the knockout mouse gained much less weight,
resulting in a phenotype more similar to the wild-type mice
on a control diet [57]. The defective mitochondria in yeast and
IP6K1 − / − MEFs, and the requirement of functional mitochondria
to synthesize the intermediates for fatty acid synthesis [84,85]
could explain the inability of IP6K1 − / − mice to gain weight,
and provides strong evidence supporting a role for inositol
pyrophosphates as regulators of metabolism at the whole organism
level. This was explored in Wundenberg and Mayr’s recent
review [65], providing a theoretical framework to understand
the possible regulatory structure behind these observations. Their
suggested cellular ‘energostat’ model, with interaction between
pyrophosphate levels and AEC, was coupled to the organismal
‘glucostat’ regulation, i.e. the established regulation between
insulin and plasma glucose concentration.
The IP6K2 − / − mouse did not have defects in insulin signalling,
implying that there is incomplete redundancy between the
isoforms [86]. This mouse is resistant to ionizing radiation, and
its fibroblasts were found to show an up-regulation in DNA
repair as well as being resistant to IFN-β (interferon-β) [86].
Conversely, overexpression of IP6K2 sensitized various human
carcinoma cells to apoptosis induced by conditions including γ irradiation, IFN-β, etoposide and cisplatin treatment [54,87,88].
Endogenous IP6K2 expression was induced by IFN-β, and IP6K
activity was increased by cisplatin treatment [54] in ovarian
cancer cells. Although individual overexpression of IP6K1, IP6K2
and IP6K3 promoted apoptosis, only knockdown of the
IP6K2 isoform in HEK (human embryonic kidney)-293 cells
increased resistance to staurospaurine [54]. This suggests that
IP6K2 may be the most important isoform in cell death
regulation by inositol pyrophosphates. The carcinogen 4-NQO (4nitroquinoline 1-oxide) was more effective at causing squamous
cell carcinoma in IP6K2 − / − mice than wild-type [86]. Protein–
protein interactions between IP6K2 and cell death/survival
regulatory proteins have also been discovered. Specifically, IP6K2
is an interacting partner of HSP90 (heat-shock protein 90) and
p53; these interactions affect the functionality of these two famous
proteins directly, with an unknown mechanism independent of
inositol pyrophosphate synthesis [89,90]. In zebrafish, which have
two known IP6K isoforms, knockdown of IP6K2 resulted in
developmental defects through alteration of Hedgehog signalling
[91].
Aging, oxidative stress and DNA repair
Metabolism and aging are intimately linked [92,93]. Levels of
both IP6 and IP7 were greatly increased in hepatocytes from
10-month-old wild-type mice compared with 2-month-old mice
[57]. Further research into possible connections between inositol
pyrophosphates and aging would be interesting. One potential link
has already been discovered via telomere length. Telomeres are
the repetitive DNA sequences that cap chromosomes, preventing
degradation. Telomeres shorten through repeated cell division,
and there is some evidence that shorter telomeres are associated
with organismal aging in mammals [94]. The yeast kcs1 strain
has been found to have slightly longer telomeres than wildtype yeast; conversely, ipk1 yeast, which have an altered
inositol pyrophosphate profile with high levels of PP-IP4 , have
375
shorter telomeres [76,77]. These effects were dependent on the
known telomere regulator Tel1, a PIKK family member and
the yeast homologue of ATM (ataxia telangiectasia mutated)
[95], suggesting that inositol pyrophosphates are physiological
inhibitors of Tel1. In support of this, yeast cell lines lacking
inositol pyrophosphates, such as kcs1, were found to be resistant
to caffeine and wortmannin, both used as inhibitors of PIKKs,
including Tel1 [76].
As well as telomere maintenance, Tel1/ATM and two other
closely related PIKKs, Mec1/ATR (ATM- and Rad3-related) and
DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) are
importantly involved in DNA damage signalling/DNA repair [96].
With Ku (XRCC5/6), DNA-PKcs forms the DNA-PK complex
required for the DNA double-strand break repair mechanism
non-homologous end-joining, the predominant double-strand
break repair mechanism used in mammalian cells. As discussed
previously, IP6 has been found to promote Ku-dependent DNA
end-joining [97,98]. It is possible that inositol pyrophosphates
can also influence this activity.
Yeast, on the other hand, are known to have a high rate
of the double-strand break repair mechanism HR (homologous
recombination) compared with mammalian cells. Several
mutations have been found that artificially increase the rate of
HR, leading to excessive deregulated recombination known as
hyper-recombination, associated with genomic instability. One
such mutation is that of Pkc1 (protein kinase C1). Knockout of
KCS1, and therefore a reduction in IP7 and IP8 , in the mutant pkc1–
4 background restores the normal rate of HR [99,100], hence the
Kcs1 name, kinase c suppressor 1. The mechanism behind this is
unclear; however, recent evidence has demonstrated that inositol
pyrophosphates can also regulate DNA repair in higher organisms
[101].
The kcs1 yeast strain was found to be more sensitive to
the DNA-damage-inducing compound phleomycin than wild-type
yeast [40]. Conversely, the kcs1 and vip1 yeast strains were
resistant to cell death and DNA mutation caused by hydrogen
peroxide treatment. This was linked to up-regulated activity
of the checkpoint kinase Rad53 (CHK2 in humans), and is
consistent with the finding that HeLa cells overexpressing IP6K2
were more sensitive to hydrogen peroxide [54]. The difference
in response between phleomycin and hydrogen peroxide in
yeast may reflect that endogenous hydrogen peroxide, normally
generated by mitochondrial activity, is likely to be decreased in
the kcs1 strain, which could alter the threshold of sensitivity for
exogeneously added hydrogen peroxide.
Interestingly, IP6K1 was found to be redox-sensitive, and
hydrogen peroxide treatment of wild-type yeast resulted in a
reduction in IP7 levels [40]. It would be very interesting to
investigate whether IP6Ks and inositol pyrophosphate synthesis is
regulated by redox mechanisms under physiological conditions,
and if ROS (reactive oxygen species) can regulate this class of
enzyme by covalently modifying their cysteine and methionine
residues. Although inositol pyrophosphate levels are necessarily
regulated by ATP availability, this would suggest further
metabolic feedback regulation.
Phosphate metabolism and polyP
Another apparently conserved link between inositol pyrophosphates and metabolism is in phosphate homoeostasis [49]. IP6K2
was first cloned as a protein that stimulated Pi uptake in Xenopus
oocytes [27,32,33]. The ability of inositol pyrophosphates to
regulate the cellular entry of Pi is conserved: the kcs1 yeast
mutant displays reduced phosphate uptake [72]. Although in
c The Authors Journal compilation c 2013 Biochemical Society
376
M. S. C. Wilson, T. M. Livermore and A. Saiardi
S. cerevisiae the Pho regulon appears to be inhibited by 1PPIP5 [60], as discussed above, alteration of phosphate metabolism
affects KCS1 transcription, thus 5PP-IP5 synthesis but not VIP1
transcription [102], indicating a complex regulatory network.
The analysis of yeast mutants unable to synthesize inositol
pyrophosphates has revealed a correlation between a lack of
inositol pyrophosphates and a dramatic decrease in the cellular
level of polyP [24,103]. The polyP polymer contains tens to
hundreds of phosphate residues linked by the same ‘high-energy’
phosphoanhydride bonds found in IP7 and ATP [47]. Many
specific biological functions have been attributed to polyP [48],
but due to its intrinsic chemical structure, this polymer mainly
represents a phosphate buffer that is synthesized and degraded as a
function of the phosphate needs of the cell. Key to ATP synthesis is
cellular phosphate homoeostasis, which may well be controlled by
inositol pyrophosphates through regulation of polyP metabolism.
Inositol pyrophosphates not only regulate phosphate metabolism
at the intracellular level, but also regulate phosphate homoeostasis
of the entire organism. In human serum, the phosphate level is
tightly regulated and its level has been genetically linked to a
single nucleotide polymorphism in the IP6K3 promoter [104].
Yeast accumulate a large amount of polyP in the vacuole,
and it is intriguing that one of the phenotypes of the kcs1
yeast is small fragmented vacuoles [34,53], suggesting vesicular
trafficking defects. Previous studies identified several trafficking
proteins as potential IP7 -binding partners, where IP6 and IP7
were found to inhibit clathrin assembly. Unfortunately, these
early binding studies were performed with non-physiological salt
concentrations [16]. Previously, a different connection between
the clathrin adaptor AP3 (adaptor protein 3) complex and IP7
has been identified in HeLa cells. AP3B1 (β-1 subunit of the
AP3 complex) is a target of pyrophosphorylation that inhibits a
protein–protein interaction between AP3B1 and the motor protein
KIF3a [74]. Release of Gag-containing virus-like particles was
used as a model to study AP3B1–KIF3a-dependent trafficking:
cells with decreased IP7 levels released more virus-like particles,
indicating that pyrophosphorylation inhibits the AP3B1–KIF3a
interaction in cells [74].
Autophagy and TOR (target of rapamycin)
Overexpression of IP6Ks in HEK-293 cells resulted in inhibition
of mTOR (mammalian TOR), and an increase in autophagy
thought to lead to cell death [105,106]. It would be interesting
to test this in more detail in the context of ATP regulation
and AMPK activity. Whether autophagy promotes survival or
cell death remains controversial. In yeast, deletion of Kcs1
attenuated autophagy caused by nitrogen starvation [107]. This
was associated with defects in autophagosome biogenesis.
Given the noted involvement of inositol pyrophosphates in
cellular metabolism, it is important to remember that TOR is
a crucial regulator of both metabolic/energy stress, via AMPK in
mammalian cells, and amino acid availability [108,109].
The inhibition of mTOR by IP6K is also interesting in the
context of ribosome biogenesis and rRNA processing. TOR
is known to up-regulate ribosome biosynthesis in response
to nutrient availability by promoting rRNA synthesis and
transcription of ribosomal proteins [110]. The nucleolus plays
an important part in this process. Notably, several targets of
pyrophosphorylation discovered so far, Nsr1, Srp40/Nopp140 and
Tcof1, are nucleolar proteins [72,73]. However, the functional
consequences of pyrophosphorylation here are unknown.
Mutation of KCS1 was found to rescue cells from a temperaturesensitive mutation of the essential nucleolar protein Rrs1 [111].
c The Authors Journal compilation c 2013 Biochemical Society
These data suggest that inositol pyrophosphates may regulate
ribosome biogenesis and thus one of the most energy-intensive
cellular processes, essential to growth and cell division [110,112].
CONCLUSION
After 20 years of research, consensus is beginning to place
inositol pyrophosphates as key regulators of cellular metabolism
(Figure 3) [57,75]. In the present review we have discussed
the major evidence for this, from the level of the cell, with the
increase in ATP and AEC in yeast and MEFs lacking inositol
pyrophosphates, to the organismal level and the impaired weight
gain and insulin hypersensitivity of mice with reduced levels of
inositol pyrophosphates. We have also attempted to re-interpret
some experimental data in light of this hypothesis. In fact, the
wide variety of processes in which inositol pyrophosphates have
been implicated, from telomere maintenance to chemotaxis, can
be best explained by these molecules acting as basic regulators
of metabolism. When considered in this way, it is clear how
inositol pyrophosphates are able to exert their influence so
widely.
This convergence of opinion [29,51,65] must drive and focus
future research. The development of new experimental tools is
essential to reveal how, exactly, this control is exerted. The
recent application of PAGE [25,113] for analysis of inositol
pyrophosphates is already facilitating new lines of inquiry. Further
innovation, with the help of our colleagues in organic chemistry,
should target production of specific inositol kinase inhibitors as
well as non-hydrolysable IP7 . Although IP6K inhibitors such
as TNP [114] are commercially available, their specificity for
IP6K over other members of the inositol phosphate kinase family,
particularly IPMK, remains in question. Better inhibitors will
allow the reversible depletion of IP7 in cells. Meanwhile, nonhydrolysable forms of IP7 [115,116], where the pyrophosphate
mimic cannot be hydrolysed, will facilitate binding studies and
help to further demonstrate the requirement of phosphotransfer
for the action of pyrophosphates.
In the 20 years since their discovery, we have amassed a good
deal of knowledge on the structure, synthesis and role of these
molecules, and yet the field remains in relative infancy. It is
worthy of note that study of the ‘PI effect’ [117], which led to the
discovery that IP3 acts as a second messenger in the mobilization
of Ca2 + stores, was already in its 30th year before that landmark
discovery was made [118]. Inositol pyrophosphates have been
known for 20 years, but we are yet to reveal the full extent of their
secrets. We look forward to the next 20 years with interest.
ACKNOWLEDGEMENTS
We thank Dr Antonella Riccio and Dr Cristina Azevedo for reading the paper before
submission and the members of the Saiardi laboratory for entertaining discussions. We
are grateful to Dr John York for sharing unpublished work.
FUNDING
This work was supported by the Medical Research Council funding of the Cell Biology
Unit, University College London, London, U.K.
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Received 22 January 2013/22 February 2013; accepted 26 February 2013
Published on the Internet 31 May 2013, doi:10.1042/BJ20130118
c The Authors Journal compilation c 2013 Biochemical Society