Roles of phosphate recognition in inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) substrate binding and activation

Article abstract of DOI:10.1074/jbc.M113.487777

Background: The mechanism of substrate recognition for IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) is unresolved. Results: Binding and activity data reveal specific roles for each phosphate of IP5. Conclusion: The phosphate profile of IP5 is mech

Full text of DOI:10.1074/jbc.M113.487777

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 37, pp. 26908–26913, September 13, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Author’s Choice
Roles of Phosphate Recognition in Inositol
1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate
Binding and Activation*
Received for publication, May 24, 2013, and in revised form, July 22, 2013 Published, JBC Papers in Press, July 24, 2013, DOI 10.1074/jbc.M113.487777
Varin Gosein‡ and Gregory J. Miller‡§¶1
From the ‡Department of Pharmacology and Therapeutics, McGill University, Montre´al, Que´bec H3G 1Y6, Canada, the §Groupe de
Recherche Axe´ sur la Structure des Prote´ines, McGill University, Montre´al, Que´bec H3G 0B1, Canada, and the ¶Department of
Chemistry, The Catholic University of America, Washington, D.C. 20064
Background: The mechanism of substrate recognition for IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) is unresolved.
Results: Binding and activity data reveal specific roles for each phosphate of IP5.
Conclusion: The phosphate profile of IP5 is mechanistically critical to IPK1 activation.
Significance: Identifying determinants of substrate specificity will aid in the design of selective inhibitors for IPK1.
Inositol phosphate kinases (IPKs) sequentially phosphorylate
inositol phosphates (IPs) to yield a group of small signaling mole-
culesinvolvedindiversecellularprocesses. IPK1(inositol1,3,4,5,6-
pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-
pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate;
however, the mechanism of IP recognition employed by IPK1 is
currently unresolved. We demonstrated previously that IPK1 pos-
sesses an unstable N-terminal lobe in the absence of IP, which led
us to propose that the phosphate profile of the IP was linked to
stabilization of IPK1. Here, we describe a systematic study to deter-
mine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol
1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation.
The 5- and 6-phosphate groups were the most important for IP
binding to IPK1, and the 1- and 3-phosphate groups were more
important for IPK1 activation than the others. Moreover, we dem-
onstrate that there are three critical residues (Arg-130, Lys-170,
and Lys-411) necessary for IPK1 activity. Arg-130 is the only sub-
strate-binding N-terminal lobe residue that can render IPK1 inac-
tive; its 1-phosphate is critical for full IPK1 activity and for stabili-
zation of the active conformation of IPK1. Taken together, our
results support the model for recognition of the IP substrate by
IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recog-
nized by the C-terminal lobe, and subsequently, (ii) the interaction
between the 1-phosphate and Arg-130 stabilizes the N-terminal
lobe and activates IPK1. This model of IP recognition, believed to
be unique among IPKs, could be exploited for selective inhibition
of IPK1 in future studies that investigate the role of higher IPs.
DNA editing and repair (2), vesicle transport (3), and ion chan-
nel regulation (4) and has been implicated in diseases such as
cancer and diabetes (5). IPs are produced by sequential phos-
phorylation of inositol 1,4,5-trisphosphate by a family of
enzymes known as IP kinases (IPKs) (1). Similarity between IPs,
which sometimes differ by only one phosphate group on the
inositol ring, demands that IPKs use mechanisms to recognize
and phosphorylate specific positions of their IP substrates while
excluding highly similar molecules. Crystal structures from
each of the IPK subfamilies have revealed that the structural
determinants for IP discrimination vary between IPKs. IP3K
(Inositol 1,4,5-trisphosphate 3-kinase) employs shape comple-
mentarity to recognize precisely positioned phosphate and
hydroxyl groups of inositol 1,4,5-trisphosphate (6). In contrast,
ITPK1 (inositol 1,3,4-trisphosphate 5/6-kinase/inositol 3,4,5,6-
tetrakisphosphate 1-kinase) discriminates among IPs using
phosphate affinity and stereochemical features to establish
contacts with phosphates that are sufficient for substrate rec-
ognition (7). Crystal structures of IPK1 (inositol 1,3,4,5,6-pen-
takisphosphate 2-kinase) in its IP substrate- and product-
bound forms reveal extensive contacts with all phosphate
groups of the bound IPs (8). These structures reveal how inosi-
tol 1,3,4,5,6-pentakisphosphate (IP5) is phosphorylated on its
axial 2Ј-hydroxyl, yielding inositol 1,2,3,4,5,6-hexakisphos-
phate, but they do not suggest a mechanism through which
IPK1 selectively recognizes IP5 as its substrate while excluding
other highly phosphorylated IPs with free axial 2Ј-hydroxyl
groups. We recently determined the crystal structure of wild-
type IPK1 in an IP-free state, which exhibited disorder within
Inositol phosphates (IPs)2 are a group of small molecules that its N-terminal lobe (N-lobe) of the kinase, centered at Arg-130
play critical roles in cellular signaling (1). IP signaling regulates (9). This IP-free structure suggests that binding of IP substrate
plays a role in stabilization of the N- and C-lobes of the kinase,
which is an important step in the activation of protein kinases
* This work was supported by Canadian Institutes of Health Research (CIHR)
(10–12).
Operating Grant MOP-93687 (to G. J. M.) and a CIHR Strategic Training Ini-
Our current objective was to define the contributions of
tiative in Chemical Biology grant (to V. G.).
Author’s Choice—Final version full access.
the individual phosphate groups of the IP to binding and to
1 To whom correspondence should be addressed: Dept. of Chemistry, The
Catholic University of America, Washington, D.C. 20064. Tel.: 202-319-
4766; Fax: 202-319-5381; E-mail: millergj@cua.edu.
2 The abbreviations used are: IP, inositol phosphate; IPK, IP kinase; IP5, inositol
1,3,4,5,6-pentakisphosphate; N-lobe, N-terminal lobe; C-lobe, C-terminal
lobe; ITC, isothermal titration calorimetry; AMP-PNP, adenosine 5Ј-(␤,␥-
imido)triphosphate; IP4, inositol tetrakisphosphate.
26908 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 37•SEPTEMBER 13, 2013

IP Recognition by IPK1
TABLE 1
recognition of bound IP as a substrate. The results demon-
strate that each phosphate group of the IP plays a different
role in binding and activation of IPK1 and that there are
three critical contacts formed between IPK1 and the IP that
mediate IPK1 activation.
Binding data of IPK1 for IP5 and IP4s
IP
N (sites)
K
M
KD
␮M
⌬H
cal/mol
Ϫ11,900 Ϫ11.4
⌬S
Ϫ1
cal/mol/degrees
IP5
0.895
1.68 ϫ 106 0.60
1,3,4,6-IP4 0.895
1,4,5,6-IP4 1.14
3,4,5,6-IP4 0.656
1,3,4,5-IP4 1.19
3.72 ϫ 104 26.88 Ϫ8720
Ϫ8.34
Ϫ2.05
1.22 ϫ 105 8.20
Ϫ7551
EXPERIMENTAL PROCEDURES
1.33 ϫ 105 7.52
Ϫ18,300 Ϫ37.9
5.49 ϫ 104 18.21 Ϫ10,140 Ϫ12.3
Generation of Alanine Mutants—Residues that interact with
IP5, either directly or through solvent molecules, were identi-
fied using previous crystal structures (9). Mutation of these res-
idues to alanine was performed by site-directed mutagenesis
using the QuikChange method (Stratagene). A pET28a vector
containing wild-type Arabidopsis thaliana IPK1 and a hexahis-
tidine tag was used as a template (a kind gift from Dr. C. A.
Brearley, University of East Anglia). All mutations were verified
by DNA sequencing.
the cell volume, K is the binding constant, and ⌬H corresponds
to the enthalpy change due to IP-IPK1 binding. The heat cor-
responding to the ith injection only, ⌬Q(i), is equal to the dif-
ference between Q(i) and Q(i Ϫ 1) and is given by Equation 2,
dVi Q͑i͒ ϩ Q͑i Ϫ 1͒
⌬Q͑i͒ ϭ Q͑i͒ ϩ
Ϫ Q͑i Ϫ 1͒ (Eq. 2)
ͩ
ͪ
Vo
2
Protein Expression and Purification—Wild-type IPK1 and
alanine mutants were expressed in BL21-AI cells (Invitrogen)
that were grown in Terrific Broth to A600 ϭ 1.5 and induced
with 0.5 mM isopropyl ␤-D-thiogalactopyranoside and 0.1% L-
arabinose at 18 °C for 20 h. Cells were lysed in 10 mM Tris-HCl
(pH 8.0), 250 mM NaCl, and 50% glycerol using a sonicator. The
supernatant was separated from the lysate by centrifugation at
45,000 ϫ g. The supernatant was then diluted 5-fold using 20
mM Tris-HCl (pH 8.0) and 500 mM NaCl, and 25 mM imidazole
was added. IPK1 was purified using nickel-nitrilotriacetic acid
beads (Thermo Scientific) in a gravity column using 4 ml of dry
beads/250 ml of culture. The beads were washed with 20 col-
umn volumes of 50 mM KPO4 (pH 8.0), 800 mM NaCl, 1% Triton
X-100, and 1.7 mM ␤-mercaptoethanol. Protein was eluted
using 250 mM imidazole in 20 mM Tris-HCl (pH 8.0) and 300
mM NaCl, and then 2 mM DTT was added to the eluate. The
protein concentration was determined by Bradford assay
(Thermo Scientific) using BSA as a standard. Protein was stored
at 4 °C and used within 72 h.
which is corrected by the injection volume (dVi) for the dis-
placed volume.
IPK1 Activity Assay—IPs (IP5, 1,3,4,5-inositol tetrakisphos-
phate (IP4), 1,4,5,6-IP4, 1,3,4,6-IP4, and 3,4,5,6-IP4) were pur-
chased from Cayman Chemical Company. A source for 1,3,5,6-
IP4 was not located. IPK1 kinase activity was assessed using the
Kinase-Glo Max luminescent kinase assay (Promega) following
the manufacturer’s instructions. Kinase reactions were per-
formed in 25-␮l volumes on black 96-well plates at 25 °C. The
reaction mixture contained 50 mM HEPES (pH 7.5), 6 mM
MgCl2, 50 mM NaCl, and 300 ␮M ATP. 25 ␮l of Kinase-Glo
reagent was added to stop the reaction, and luminescence was
measured after 20 min on a Berthold Orion II microplate lumi-
nometer. Initially, 80 ␮M IP was used, and the amount of
enzyme was varied to determine conditions in which product
formation was linear over 30 min. Subsequently, an array of
reactions with varying concentrations of IP (20, 40, 60, 80, 100,
120, and 140 ␮M) stopped at various time points (2, 5, 10, 20,
and 30 min) were performed in triplicate. The rate of product
formation versus IP concentration was plotted and fitted to the
Michaelis-Menten equation using nonlinear regression to
determine Km and Vmax (GraphPad Software). The kcat values
were calculated using the equation kcat ϭ Vmax/[E], where [E] is
the micromolar enzyme concentration.
Isothermal Titration Calorimetry (ITC)—Experiments were
performed on a MicroCal iTC200 titration calorimeter (GE
Healthcare). Wild-type IPK1 was purified and dialyzed into
ITC buffer containing 50 mM HEPES (pH 7.5), 6 mM MgCl2, 150
mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine (pH 7.0).
After protein dialysis was complete, dialysis buffer was used to
dissolve the ligands, IP, and AMP-PNP (Jena Bioscience). Titra-
tion experiments were performed at 25 °C with 100 ␮M IPK1
and 1 mM AMP-PNP in the cell and 1–2 mM IP in the syringe to
ensure a final IP:IPK1 molar ratio of at least 2:1. Titration exper-
iments were performed at least twice for each IP, and one set
was chosen to represent data. Calorimetric data were analyzed
using Origin 7.0 (MicroCal). Data were fitted with a one-site
model using Equation 1,
IPK1 Alanine Mutant Activity Assay—Initially, 150 ng of
each mutant was tested for kinase activity with 80 ␮M IP5 after
30 min. Mutants that exhibited little or no activity were retested
using 750 ng of enzyme. Active mutants were further charac-
terized for their kinetic parameters with IP5 as a substrate using
the abovementioned approach.
RESULTS
5- and 6-Phosphates Are Important for Binding—To deter-
mine the role of phosphates at each position of the inositol ring,
we measured the effect on binding affinity when using IPs lack-
ing a phosphate group at the 1-, 3-, 5-, or 6-position. Using ITC,
we obtained KD values for each IP4 and IP5 (Table 1). As
expected, IP5, the native substrate for IPK1, displayed the high-
est binding affinity, with KD ϭ 0.60 ␮M. IP4s exhibited a range of
binding affinities. The KD values of 1,4,5,6-IP4 and 3,4,5,6-IP4
1
Xt
1
ͩ
Q͑i͒ ϭ nPt⌬HV 1 ϩ
ϩ
Ϫ
2
nPt nKPt
1
2
2
Xt
1
4Xt
ͩͩ
ͪ ͪ ͪ
1 ϩ
ϩ
Ϫ
(Eq. 1)
nPt nKPt
nPt
where n is the number of IP-binding sites on IPK1, Pt is the total were 13-fold higher than that of IP5, whereas the KD values of
concentration of IPK1, Xt is the total concentration of IP, V is 1,3,4,6-IP4 and 1,3,4,5-IP4 were at least 30-fold higher. These
SEPTEMBER 13, 2013•VOLUME 288•NUMBER 37
JOURNAL OF BIOLOGICAL CHEMISTRY 26909

IP Recognition by IPK1
TABLE 3
Kinetic parameters of IPK1 alanine mutants for IP5
Data represent the mean Ϯ S.D. of triplicate experiments. The last column indicates
the IP phosphate that interacts with the mutant side chain, either directly or indi-
rectly, through ordered water molecules. ND, no activity detected.
Mutant
Km
␮M
kcat
min
PO4 interaction
Ϫ1
R45A
54.28 Ϯ 9.07
ND
27.16 Ϯ 1.79
3
R130A
K168A
K170A
R192A
H196A
K200A
N238A
N239A
D368A
K411A
R415A
Y419A
ND
1
ND
ND
2
ND
ND
5, 6
5
59.35 Ϯ 13.41
43.48 Ϯ 15.62
39.79 Ϯ 19.52
33.72 Ϯ 7.918
83.30 Ϯ 9.53
ND
22.01 Ϯ 2.05
34.12 Ϯ 4.33
16.74 Ϯ 2.77
14.74 Ϯ 1.07
15.89 Ϯ 0.87
ND
5
6
6
6
2
FIGURE 1. Kinetic analysis of the kinase activity of IPK1 for IP5 and IP4s.
IPK1 kinase activity was assessed using a luminescence-based assay. The rate
of product formation versus IP concentration was plotted and fitted to the
Michaelis-Menten equation. Each point represents the mean Ϯ S.D. of tripli-
cate experiments. F, IP5; f, 1,3,4,6-IP4; ࡗ, 3,4,5,6-IP4; Œ, 1,4,5,6-IP4. enz,
enzyme.
ND
ND
3, 4
3, 4
4
62.27 Ϯ 21.89
76.86 Ϯ 16.94
38.71 Ϯ 5.73
41.62 Ϯ 4.25
between mutants, we determined kcat values for each mutant.
For wild-type IPK1, kcat ϭ 44.02 Ϯ 3.19 nmol/min. Mutation of
Arg-130, the only residue that interacts with the 1-phosphate of
the IP, resulted in no detectable activity. Mutations of Lys-168
and Asp-368, which mediate phosphoryl transfer from ATP to
the 2Ј-hydroxyl, also exhibited no activity, as expected. The
R45A mutant, which abolished the contact with the 3-phos-
TABLE 2
Kinetic parameters of IPK1 for IP5 and IP4s
Data represent the mean Ϯ S.D. of triplicate experiments. ND, no activity detected.
IP
Km
␮M
kcat
min
44.02 Ϯ 3.19
27.48 Ϯ 4.00
6.60 Ϯ 0.66
6.30 Ϯ 0.68
ND
Ϫ1
IP5
63.05 Ϯ 10.77
66.81 Ϯ 22.37
55.99 Ϯ 13.95
47.54 Ϯ 13.94
ND
1,3,4,6-IP4
1,4,5,6-IP4
3,4,5,6-IP4
1,3,4,5-IP4
phate, displayed a 40% decrease in kinase activity (kcat
ϭ
27.16 Ϯ 1.79 nmol/min), whereas the R415A and Y419A
mutants, which abolished contacts with the 4-phosphate, both
displayed activity equivalent to wild-type IPK1. In contrast, the
K411A mutant, which abolished contacts with both the 3- and
4-phosphates, showed no activity. The R192A mutant dis-
played a modest decrease in kinase activity (kcat ϭ 22.01 Ϯ 2.05
nmol/min), and the H196A mutant had no effect on kinase
activity (kcat ϭ 34.12 Ϯ 4.33 nmol/min), demonstrating that
interactions with the 5-position have modest impact on cata-
lytic activity. The K170A mutant, which abolished interactions
with both the 5- and 6-phosphates, showed no activity. Finally,
the K200A, N238A, and N239A mutants, which eliminated
contacts with the 6-phosphate, displayed reduced activity com-
pared with wild-type IPK1. Fig. 2 summarizes the effect of ala-
nine mutations in the inositide-binding site on IPK1 activity.
Here, we observed that the residues that interact with more
than one phosphate play important roles in substrate recogni-
tion, whereas most residues that interact with a single phos-
phate play lesser roles in this process.
results indicate that different phosphate groups have varying
contributions to the binding affinity of IP5 for IPK1. Compari-
son of the IP4 KD:IP5 KD ratios revealed that the 5- and 6-phos-
phates contributed the most to the binding affinity of the IP, as
the absence of either phosphate group dramatically increased
the KD (Table 1).
1- and 3-Phosphates Are Important for Substrate Recognition—
We tested the kinase activity of IPK1 for IP5 and IP4s using a
luminescence-based assay to determine which phosphates
identify an IP as a substrate for IPK1 (Fig. 1 and Table 2). The
kinase activity of IPK1 was maximal in the presence of IP5, with
k
cat ϭ 44.02 Ϯ 3.19 nmol/min. With 1,3,4,6-IP4, an IP lacking
the 5-phosphate, there was a modest decrease in kinase activity,
with kcat ϭ 27.48 Ϯ 4.00 nmol/min. In contrast, IPs lacking 1-
and 3-phosphate groups exhibited a substantial 85% decrease in
activity compared with IP5. No activity was detected when
1,3,4,5-IP4 was used as a substrate, suggesting that the 6-phos-
phate may also be important for activation. These results indi-
cate that the 1- and 3-phosphates are important for the IP to be
DISCUSSION
Roles of Different Phosphate Groups: Binding versus Activation—
recognized as a substrate by IPK1. We also observed that the KD Here, we performed a systematic study to identify the relative
values for IPs lacking phosphates varied considerably, whereas contributions of the 1-, 3-, 5-, and 6-phosphates to IP5 binding
the Km for each IP remained nearly constant (Table 2). This affinity and recognition as an IPK1 substrate. Initially, we used
disconnect suggests that the kinetic parameters of ligand bind- ITC to determine the binding affinity of IPK1 for IP5 and for a
ing or catalysis change along with the binding affinity, but we set of IP4s, each lacking a single phosphate group. We observed
cannot define with the current set of assays how they change.
a spectrum of affinities for these differently phosphorylated IP4
Alanine Mutants Identify Active Site Residues Critical for molecules, indicating that phosphates contribute differently to
IPK1 Activity—To identify the contacts between IPK1 and IP binding. IPs lacking the 5- or 6-phosphate displayed the lowest
that are essential for activity, we mutated to alanine those IPK1 binding affinity for IPK1, whereas IPs lacking the 1- or 3-phos-
residues that interact with the IP and tested these mutants for phate displayed only moderately decreased binding affinity
activity for IP5 (Table 3). To compare the kinase activity (Table 1). The structure of nucleotide-bound IPK1 (Protein
26910 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 37•SEPTEMBER 13, 2013

IP Recognition by IPK1
substrate to levels below the detection limit of our assay. The
KD values for 1,3,4,5-IP4 and 1,3,4,6-IP4 were similar; however,
1,3,4,6-IP4 could be used as a substrate, whereas 1,3,4,5-IP4
could not. This indicates that the decreased binding affinity of
1,3,4,5-IP4 for IPK1 was not by itself the underlying factor for its
inability to be used as a substrate (Table 1). The 6-phosphate-
binding site plays a key role in IPK1 activation by preventing
clasp formation (a critical step in the IPK1 catalytic cycle) in the
absence of IP substrate. Binding of the 6-phosphate to Lys-200
disrupts the interaction between Lys-200 and Glu-255, and this
newly freed Glu-255 binds to Trp-129, thereby promoting clasp
formation between the L3 loop and ␣6 helix (13). However, the
K200A mutant, as well as other mutants that disrupted interac-
tion with the 6-phosphate (K170A, N238A, and N239A), did
not show any activity for 1,3,4,5-IP4, so intramolecular changes
in the 6-phosphate-binding site are not essential for IPK1 acti-
vation (data not shown). It is possible that 1,3,4,5-IP4 adopts
alternative binding orientations in which the 2Ј-hydroxyl is not
accessible for phosphorylation (7, 15). Further experimentation
will be required to ascertain why 1,3,4,5-IP4 displays no activity.
Critical Roles of Arg-130 and 1-Phosphate in IPK1 Activation—
On the basis of previous crystal structures, we mutated each
substrate-binding residue to alanine to determine the critical
contacts between IPK1 and the IP. Each mutation abolished an
interaction with one or two phosphates (Table 3), and we deter-
mined the Km and kcat for each mutant in the presence of IP5.
FIGURE 2. Structural representation of kinetic parameters of alanine
mutants. IP5 is shown in yellow stick form. The side chains of IP-binding res-
idues are shown in stick form and colored according to alanine mutant kcat
.
Maroon indicates no activity. Darker green shades indicate reduced activity
compared with wild-type IPK1. Green indicates equivalent activity to wild-
type IPK1. Side chains are grouped according to bound phosphate, overlaid
with colored arcs (red, N-lobe; blue, C-lobe).
Data Bank code 3UDS) revealed the N-lobe to be unstable com- The impacts of the mutations and the activities of the IP4s were
pared with the IP-bound state, and in a recent structure of IPK1 largely symmetric; abolishing single contacts with a phosphate
engineered to crystallize in the absence of IP (Protein Data Bank group through mutation had a similar impact as removing the
code 4AXC), the N-lobe was too far away from the C-lobe to phosphate from the substrate. A notable difference between the
form a complete inositide-binding pocket (9, 13). In both wild- IPK1 mutant and IP4 data was the asymmetric impact of abol-
type and mutant structures, the N-lobe fails to assemble into ishing the interaction with the 1-phosphate. Given that Arg-
the active conformation. Our ITC binding data, which indicate 130, located on the N-lobe, is the only residue that interacts
that the C-lobe-binding 5- and 6-phosphates contribute sub- with the 1-phosphate, we expected that an IP lacking the
stantially more to binding, are consistent with the C-lobe play- 1-phosphate would correspond with the activity of this mutant.
ing a dominant role in substrate recruitment. This collection of However, the R130A mutant displayed no detectable activity,
structures and the binding data support the model that sub- whereas 3,4,5,6-IP4 could be used by the wild-type enzyme,
strate recruitment likely occurs though the stable C-lobe, which albeit with low activity (Tables 2 and 3). Structures of IPK1 in
comprises half of the IP-binding site. To complete assembly of complex with IP show that Arg-130 forms a bond with Gly-254,
the IP-binding site, IP must bind to both the N- and C-lobes, establishing an interaction between the L3 loop and ␣6 helix
thereby coupling binding of the substrate to stabilization of the and promoting clasp formation (Fig. 3) (9). This interaction,
kinase.
and therefore clasp formation, cannot occur in the R130A
We further investigated the contribution of each phosphate mutant, rendering IPK1 inactive. Transient clasp formation can
group of the IP to its recognition as a substrate and to activation occur in wild-type IPK1, even in the absence of a 1-phosphate,
of IPK1. We determined the Km and kcat of IPK1 in the presence which allows the use of 3,4,5,6-IP4 as a substrate with low activ-
of IP5 and our series of IP4s. These data indicate that the kcat for ity. The 1-phosphate interaction with Arg-130 is conducive to
IPK1 is substantially decreased for IPs lacking the 1- or 3-phos- clasp formation, thereby rendering IPK1 fully active (Fig. 3).
phate compared with IP5 and 1,3,4,6-IP4 (Table 2). Preliminary Thus, the 1-phosphate stabilizes the active conformation of
studies of IPK1 substrate specificity also revealed the use of IPK1.
1,3,4,6-IP4 as a substrate, but not 3,4,5,6-IP4; however a kinetic
One Critical Residue at Each Phosphate-binding Site—There
analysis of IPK1 with each IP4 was not performed (14). Our are 13 residues that interact with the IP in the IPK1 active site,
observations are consistent with the 1- and 3-phosphates stabi- either directly or indirectly through ordered water molecules,
lizing the bilobed structure of IPK1, thereby promoting its acti- and each of these residues results in a bond to a phosphate
vation though recruitment of the N-lobe using a mechanism group. For each phosphate of the IP, there is a contact residue
similar to that reported for protein kinases (12).
that plays a critical role, and mutation of that residue abolishes
IPK1 was unable to use 1,3,4,5-IP4 as a substrate, which sug- activity. As discussed above, Arg-130 interacts with the 1-phos-
gests that the 6-position may play a dual role in both IP binding phate of the substrate and is a primary contact between the IP
and activation; abolishing both functions decreased its use as a and the kinase N-lobe. Arg-130 is essential due to its structural
SEPTEMBER 13, 2013•VOLUME 288•NUMBER 37
JOURNAL OF BIOLOGICAL CHEMISTRY 26911

IP Recognition by IPK1
and Lys-411 impact binding affinity to the extent that substrate
recognition cannot occur or if these residues play structural
roles in shaping the binding site into an active conformation, as
Arg-130 does, remains to be determined.
Model of IP-induced Stabilization as a Mechanism of Selec-
tivity of IPs—Our previous crystal structures revealed that IPK1
possesses a stable C-lobe and a destabilized N-lobe in the
absence of IP, such that the inositide-binding pocket remains
partially formed (9). We proposed a model wherein IPK1 links
its interactions with IP substrate phosphate groups to stabiliza-
tion of the N- and C-lobes and kinase activation. The stability of
the C-lobe in the absence of IPs suggested that the roles of 4-, 5-,
and 6-phosphates were to mediate the initial contact of the IP
with IPK1. Our present study reveals that the 5- and 6-phos-
phates impact binding affinity more than the 1- and 3-phos-
phates. Our model also proposed that the 1- and 3-phosphates
were required for N-lobe stabilization, as they act to bridge the
N-lobe with the C-lobe. In our present study, IPK1 displayed
substantially lower kcat values with 3,4,5,6-IP4 and 1,4,5,6-IP4
than with IP5 or an IP4 lacking the 5-phosphate. IP-free crystal
structures of IPK1 reveal that the destabilization of the
N-lobe is centered at Arg-130, which directly binds to the
1-phosphate of the IP substrate (9). The mutation of R130A
impaired IPK1 activity, suggesting that the N-lobe is
required to be stabilized for IPK1 activation, a key feature of
kinase activation (12). In short, our study reveals specific
roles for each of the IP phosphates, linking IPK1 substrate
specificity to IPK1 stability.
FIGURE 3. Clasp formation between the ␣6 helix and L3 loop. Interactions
between Arg-130 (magenta stick form) and Gly-254 (cyan stick form) mediate
clasp formation between the L3 loop (cyan) and ␣6 helix (magenta) of the
C-lobe (blue) and of the N-lobe (red), respectively. Arg-130 interacts with the
1-phosphate of the IP, which is conducive to clasp formation. This image
was created using PyMOL and a product-bound structure of IPK1 (Protein
Data Bank code 3UDZ). ADP (green) and inositol 1,2,3,4,5,6-hexakisphos-
phate (IP6; yellow) are shown in stick form. Dashed lines indicate hydrogen
bonds.
role in stabilizing the active state and not exclusively for its role
Conclusions—In this work, we have demonstrated that the
in binding the 1-phosphate, as 3,4,5,6-IP4 can bind and be rec- phosphate profile of IP5 is mechanistically linked to IPK1 acti-
ognized as a substrate. An IP lacking the 3-phosphate also dis- vation. We have identified phosphates at the 1-, 3-, and 6-posi-
played low activity; however, the R45A mutation, which dis- tions as the most important for activation of IPK1, whereas
rupted the second contact between the IP and N-lobe, phosphates at the 5- and 6-positions are more important for
moderately impaired activity (Table 3). Thus, it appears that binding than those at the 1- and 3-positions. Identification of
N-lobe interaction with IP is mediated primarily through Arg- the roles of the phosphates of the IP supports our proposed
130 and the 1-phosphate. Mutation of Lys-411, which interacts model of IPK1 substrate specificity and may provide a basis for
with both the 3- and 4-phosphates, was critical for activity, yet selective targeting of IPK1, as similarity among IP substrates
mutations of Arg-415 and Tyr-419, which mediate other inter- continues to hinder development of specific inhibitors for IPKs.
actions with the 4-phosphate, did not markedly affect IPK1 Inhibition of IPK1 would prove valuable in the investigation of
activity, nor did R45A, which is the second contact with the the roles of higher IPs whose production is dependent on ino-
3-phosphate, as described above (Table 3). Both the 5- and sitol 1,2,3,4,5,6-hexakisphosphate, the product of IPK1, as well
6-phosphates bind to Lys-170, which is critical for activity; as functional roles of IPK1 in mammals.
however, other 5-phosphate interactions with Arg-192 and
Acknowledgments—We thank Dr. C. A. Brearley for the gift of the
pET28-AtIPK1 vector, Dr. Guennadi Kozlov (McGill University) for
technical assistance with ITC, Dr. Dan Bernard (McGill University)
for access to the luminometer used in enzyme assays, Dr. Hatem
Dokainish (McGill University) for helpful discussions, and Anne W.
Coventry for thoughtful reading of the manuscript.
His-196 and other 6-phosphate interactions with Lys-200, Asn-
238, and Asn-239 are dispensable for activity. Wild-type IPK1
displayed activity with IPs lacking the 3- or 5-phosphate, yet
mutations of Lys-170 and Lys-411 rendered IPK1 inactive,
likely due to the fact that these residues bind more than one
phosphate (Tables 2 and 3). Sequence alignments of IPK1 reveal
that Arg-45, Arg-415, and Tyr-419 are the only inositide-bind-
ing residues that are not conserved among plant, human, rat,
and yeast (8). Accordingly, R45A, R415A, and Y419A mutations
retained at least 60% of wild-type activity. The conservation of
Arg-130, but not Arg-45, suggests that N-lobe contact with the
IP is mediated primarily through the 1-phosphate. In summary,
our data indicate that each phosphate has a critical residue in
the active site without which the enzyme cannot function, nota-
bly Arg-130, Lys-170, and Lys-411 (Table 3). Whether Lys-170
REFERENCES
1. Irvine, R. F., and Schell, M. J. (2001) Back in the water: the return of the
inositol phosphates. Nat. Rev. Mol. Cell Biol. 2, 327–338
2. Hanakahi, L. A., Bartlet-Jones, M., Chappell, C., Pappin, D., and West, S. C.
(2000) Binding of inositol phosphate to DNA-PK and stimulation of dou-
ble-strand break repair. Cell 102, 721–729
3. Hilton, J. M., Plomann, M., Ritter, B., Modregger, J., Freeman, H. N., Falck,
J. R., Krishna, U. M., and Tobin, A. B. (2001) Phosphorylation of a synaptic
vesicle-associated protein by an inositol hexakisphosphate-regulated pro-
26912 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 37•SEPTEMBER 13, 2013

IP Recognition by IPK1
tein kinase. J. Biol. Chem. 276, 16341–16347
phate 2-kinase and its role in substrate specificity. Protein Sci. 21,
737–742
10. Knighton, D. R., Zheng, J. H., Ten Eyck, L. F., Xuong, N. H., Taylor, S. S.,
and Sowadski, J. M. (1991) Structure of a peptide inhibitor bound to the
catalytic subunit of cyclic adenosine monophosphate-dependent protein
kinase. Science 253, 414–420
4. Vajanaphanich, M., Schultz, C., Rudolf, M. T., Wasserman, M., Enyedi, P.,
Craxton, A., Shears, S. B., Tsien, R. Y., Barrett, K. E., and Traynor-Kaplan,
A. (1994) Long-term uncoupling of chloride secretion from intracellular
calcium levels by Ins(3,4,5,6)P4. Nature 371, 711–714
5. Shi, Y., Azab, A. N., Thompson, M. N., and Greenberg, M. L. (2006) Ino-
sitol phosphates and phosphoinositides in health and disease. Subcell. 11. Ozkirimli, E., and Post, C. B. (2006) Src kinase activation: a switched elec-
Biochem. 39, 265–292
trostatic network. Protein Sci. 15, 1051–1062
6. Gonza´lez, B., Schell, M. J., Letcher, A. J., Veprintsev, D. B., Irvine, R. F., and
Williams, R. L. (2004) Structure of a human inositol 1,4,5-trisphosphate
12. Sicheri, F., Moarefi, I., and Kuriyan, J. (1997) Crystal structure of the Src
family tyrosine kinase Hck. Nature 385, 602–609
3-kinase: substrate binding reveals why it is not a phosphoinositide 3-ki- 13. Ban˜os-Sanz, J. I., Sanz-Aparicio, J., Whitfield, H., Hamilton, C., Brearley,
nase. Mol. Cell 15, 689–701
C. A., and Gonza´lez, B. (2012) Conformational changes in inositol
1,3,4,5,6-pentakisphosphate 2-kinase upon substrate binding. Role of N-
terminal lobe and enantiomeric substrate preference. J. Biol. Chem. 287,
29237–29249
7. Miller, G. J., Wilson, M. P., Majerus, P. W., and Hurley, J. H. (2005)
Specificity determinants in inositol polyphosphate synthesis: crystal
structure of inositol 1,3,4-trisphosphate 5/6-kinase. Mol. Cell 18,
201–212
14. Sweetman, D., Johnson, S., Caddick, S. E., Hanke, D. E., and Brearley, C. A.
(2006) Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakispho-
sphate 2-kinase (AtIPK1). Biochem. J. 394, 95–103
8. Gonza´lez, B., Ban˜os-Sanz, J. I., Villate, M., Brearley, C. A., and Sanz-Apa-
ricio, J. (2010) Inositol 1,3,4,5,6-pentakisphosphate 2-kinase is a distant
IPK member with a singular inositide binding site for axial 2-OH recog- 15. Chamberlain, P. P., Qian, X., Stiles, A. R., Cho, J., Jones, D. H., Lesley, S. A.,
nition. Proc. Natl. Acad. Sci. U.S.A. 107, 9608–9613
Grabau, E. A., Shears, S. B., and Spraggon, G. (2007) Integration of inositol
phosphate signaling pathways via human ITPK1. J. Biol. Chem. 282,
28117–28125
9. Gosein, V., Leung, T. F., Krajden, O., and Miller, G. J. (2012) Inositol
phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphos-
SEPTEMBER 13, 2013•VOLUME 288•NUMBER 37
JOURNAL OF BIOLOGICAL CHEMISTRY 26913

Enzymology:
Roles of Phosphate Recognition in Inositol
1,3,4,5,6-Pentakisphosphate 2-Kinase
(IPK1) Substrate Binding and Activation
Varin Gosein and Gregory J. Miller
J. Biol. Chem. 2013, 288:26908-26913.
doi: 10.1074/jbc.M113.487777 originally published online July 24, 2013
Access the most updated version of this article at doi: 10.1074/jbc.M113.487777
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
This article cites 15 references, 5 of which can be accessed free at
http://www.jbc.org/content/288/37/26908.full.html#ref-list-1