Inositol polyphosphate multikinase is a nuclear
PI3-kinase with transcriptional regulatory activity
Adam C. Resnick*, Adele M. Snowman*, Bingnan N. Kang†, K. Joseph Hurt*, Solomon H. Snyder*†‡§,
and Adolfo Saiardi*¶§
Departments of *Neuroscience,†Pharmacology, and‡Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine,
725 North Wolfe Street, Baltimore, MD 21205
Contributed by Solomon H. Snyder, July 21, 2005
Phosphatidylinositol 3,4,5-trisphosphate is a major intracellular
messenger molecule thought to be formed almost exclusively by
cytosolic, wortmannin-inhibited phosphoinositide 3-kinase family
members. Inositol polyphosphate multikinase was identified as an
enzyme that generates a series of water-soluble inositol phos-
phates. We now report the robust, physiologic, and evolutionarily
conserved phosphoinositide 3-kinase activity of inositol polypho-
sphate multikinase, which is localized to nuclei and unaffected by
wortmannin. In yeast, this inositol lipid kinase activity physiolog-
ically regulates transcription.
lipids ? nucleus ? wortmannin
phate substituents on the myo-inositol ring (1). Inositol 1,4,5-
releases intracellular calcium, is generated by the hydrolysis of
the lipid inositol phosphatidylinositol 4,5-bisphosphate
[PI(4,5)P2] by phospholipase C (PLC) (2) and can be sequen-
tially phosphorylated by inositol phosphate kinases (3). A re-
cently identified family of inositol polyphosphate kinases (IPKs;
PFAM accession no. PF03770) includes the inositol
hexakisphosphate kinases (IP6Ks), the IP3-3 kinases, and a third
member that we designated inositol polyphosphate multikinase
(IPMK, also designated Ipk2) because of its ability to phosphor-
ylate multiple sites on the inositol phosphate ring (4–6). Se-
quential phosphorylation of IP3by IPMK ultimately leads to the
production of inositol 1,3,4,5,6-pentakisphosphate (IP5) (Fig. 1).
Yeast mutants of IMPK display transcriptional regulation de-
fects in response to nutrients, as well as defects in sporulation,
mating, and salt stress tolerance (7).
Besides generating IP3, PI(4,5)P2is a substrate for the pre-
dominantly cytosolic lipid inositol kinases designated phospho-
inositide 3-kinases (PI3Ks) which phosphorylate the D-3 posi-
tion of inositol lipids, often in response to growth factor receptor
stimulation (8, 9). The formation of phosphatidylinositol 3,4,5-
trisphosphate [PI(3,4,5)P3] at the plasma membrane by class I
PI3Ks is the rate-limiting step in multiple pathways that regulate
cell migration, growth, proliferation, and survival (8). We now
report that IPMK is a robust, physiologic nuclear PI3K that
regulates transcription (Fig. 1).
ater-soluble inositol phosphates comprise a family of
signaling molecules with various permutations of phos-
Materials and Methods
Lipid Kinase Assays. Lipid inositol substrates were dried under a
stream of nitrogen gas and resuspended via sonication in a
carrier of phosphatidylserine, 20 mM Hepes (pH 7.4), and 1 mM
EDTA. Alternatively, lipid inositol substrates were resuspended
in 20 mM Hepes (pH 7.4), 1 mM EDTA, and 0.5% deoxycholate.
Both methods yielded similar results. Kinase reactions were
performed in a total volume of 50 ?l containing 10 ?l of lipid
resuspension providing a final concentration of 0.03 mg?ml
purified?synthetic lipid inositols (Sigma, Calbiochem, Avanti
Polar Lipids) or at a final concentration of 0.2 mg?ml for Folch
bovine brain extracts (Sigma). Kinase reaction buffer consisted
of 20 mM Hepes (pH 7.4), 6 mM MgCl2, and 10 ?Ci of
[?-32P]ATP (PerkinElmer–NEN, 6,000 mCi?mmol; 1 Ci ? 37
GBq) in a carrier of 100 ?M unlabeled ATP. Enzyme reactions
were incubated either 30°C (yeast IPMK) or 37°C (rat IPMK and
p110?) for 15 min. Enzyme concentrations typically ranged
between 10 and 50 ng of bacterially expressed and purified
His-?GST-tagged IPMK or His-tagged human p110? (Alexis
Biochemicals). Enzymatic comparisons were made by using
equal molar enzyme concentrations. Kinase reactions were
stopped with 90 ?l of 1 M HCl?methanol (1:1 by volume). Lipids
were extracted twice with 100 ?l of choloroform and resolved on
silica gel 60 TLC plates in a solvent system consisting water?n-
propanol?glacial acetic acid (34:65:1, by volume). Alternatively,
extracted lipids were deacylated as described (10) and separated
Characterization of Enzyme Kinetics. Kmand Vmaxdeterminations
were performed by using a maximum of 100 ?M PI(4,5)P2
(Avanti Polar Lipids) or serial dilutions of the lipid resuspended
in 20 mM Hepes (pH 7.4), 1 mM EDTA, and 0.5% deoxycholate.
Reactions were performed in triplicate as described above.
Extracted, radiolabeled lipids were counted, and rate calcula-
tions were fitted to Michaelis–Menten equations by using
SIGMAPLOT. Competition studies using IP3 or inositol 1,3,4,6-
tetrakisphosphate (A.G. Scientific) were performed in the pres-
ence of 100 ?M PI(4,5)P2and increasing concentrations of the
indicated soluble inositol.
HPLC Analysis of Soluble and Lipid Inositol. Isolated glycerophos-
phoinositides were resuspended in 1 mM EDTA, and ?2 ? 106
cpm were resolved by using anion exchange chromatography
with a Whatman PartiSphere SAX (4.6 ? 250 mm) column. The
column was eluted with a gradient generated by mixing 1 mM
EDTA and buffer B [1 mM EDTA?1.3 M (NH4)2HPO4, pH 3.8]
in one of two methods: (i) 0–5 min, 0% B; 5–105 min, 0–100%
B; 85–105 min, 50%–100% B; 105–120 min, 100% B. Spectro-
photometric monitoring of AMP, ADP, and ATP elution times
and authentic deacylated [3H]PI(4,5)P2(PerkinElmer NEN) or
p110?-generated [32P]PI(3,4,5)P3and [32P]PI(3,4)P2were used
as standards. Singly phosphorylated glycerophosphoinositides
species eluted between AMP and ADP, whereas glycerophos-
phoinositide bisphosphates such as gPI(4,5)P2eluted between
ADP and ATP; gPI(3,4,5)P3eluted after ATP. Fractions (1 ml)
Abbreviations: IP3, inositol 1,4,5-trisphosphate; PI(4,5)P2, inositol phosphatidylinositol 4,5-
bisphosphate; PLC, phospholipase C; IPK, inositol polyphosphate kinase; IPMK, inositol
polyphosphate multikinase; PI3K, phosphoinositide 3-kinase; PI(3,4,5)P3, phosphatidylino-
¶Present address: Medical Research Council (MRC) Cell Biology Unit and Laboratory for
Molecular Cell Biology, Department of Biochemistry and Molecular Biology, University
College London, Gower Street, London WC1E 6BT, United Kingdom.
§To whom correspondence may be addressed. E-mail: email@example.com or ssnyder@
© 2005 by The National Academy of Sciences of the USA
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September 6, 2005 ?
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were collected and counted by using 4 ml of Ultima-Flo AP
LCS-mixture (Packard). Extraction and analyses of soluble
inositol lipids were performed as described (11).
Lipid Phosphatase Reactions. Lipid phosphatase reactions were
performed as per manufacturers’ recommendations using re-
(Upstate Biotechnology) and putative [32P]PI(3,4,5)P3 gener-
ated in vitro as described above. Extracted lipids were resolved
by TLC and analyzed by autoradiography.
Transfections. HEK293T and Cos-7 (American Type Culture
Collection) cells were transfected by using Lipofectamine 2000
(Invitrogen) as per the manufacturer’s recommendation.
[3H]Inositol Labeling of Transfected Cells. Transfected HEK293T
and Cos-7 cells were labeled for 48 h with 10 ?Ci?ml
(PerkinElmer NEN, 25 Ci?mmol) in inositol-free DMEM (Spe-
cialty Media). Lipid inositols were extracted as described (10)
and analyzed by HPLC as described above.
Primary Cultures. Primary cortical and hippocampal cultures were
established as described (12). Immunostaining and activity as-
says were performed on 3- to 5-day-old cultures. Hepatocyte
cultures were established by using GIBCO hepatocyte products
(Invitrogen) as per the manufacturer’s recommendations.
Immunostaining. Transfected cells and primary cultures were
fixed in 4% paraformaldehyde for 30 min at 4°C. Cells were
washed three times in Tris-buffered saline (TBS) and perme-
abilized in TBS containing 10% goat serum (Vector Laborato-
ries) and 0.5% Triton X-100 for 1 h at room temperature.
Primary antibodies were incubated overnight in TBS containing
10% goat serum. Primary antibodies were used at the following
concentrations: anti-HA (Covance), 1:5,000; anti-?III-tubulin
(Promega), 1:2,000; anti-PI(3,4,5)P3(13) (Echelon), 1:200; anti-
neurofilament (Developmental Studies Hybridoma Bank),
1:100; anti-IPMK, 1:1,500. After primary antibody incubation,
cells were washed five times in TBS containing 10% goat serum.
Alexa Fluor 488 or Alexa Fluor 568 fluorescent secondaries
(1:4,000) (Molecular Probes) were applied for 5 h at room
temperature in TBS containing 10% goat serum. Cells were
washed overnight in PBS. Identification of nuclei was performed
by using Hoechst dye 33258 (Molecular Probes). Polyclonal
anti-IPMK antibodies were generated in rabbits by using full-
length bacterially expressed rat IPMK; IPMK-specific antibodies
were affinity purified by using Affigel-crosslinked (Bio-Rad) rat
IPMK. Confocal images were obtained on a PerkinElmer
UltraVIEW Spinning Disk Confocal microscope.
Nuclear Extraction and Immunoprecipitations. IPMK immunprec-
ipitations were performed by using 1 mg of cell extract from
isolated nuclei prepared as described (14). Nuclei were lysed in
20 mM Hepes, pH 7.4?1.5 mM MgCl?400 mM NaCl?1% Non-
idet P-40?0.2 mM EDTA, and protease inhibitor mixture
(Sigma). Alternatively, nuclear lysates were prepared by using
NE-PER Nuclear extraction reagent (Pierce). Lysates were
incubated with 2 ?g of anti-IPMK antibody and 30 ?l of protein
at 4°C. Immunoprecipitates were washed five times with a wash
buffer consisting of 20 mM Hepes (pH 7.4), 500 mM NaCl, and
1% Nonidet P-40 followed by two washes in 1? kinase reaction
buffer. Reactions were performed on immunoprecipitates as
described above, but incubated for 1 h as IPMK-specific anti-
bodies significantly inhibit enzymatic activity. PI3K p85 immu-
noprecipitation was performed as per manufacturer’s protocol
(Upstate Biotechnology), typically from 1 mg of cell lysate.
Control immunoprecipitates were performed with equal con-
centrations of purified rabbit IgG (Vector Laboratories).
Akt Actvation Assays. Transfected HEK293T cells were treated
with IGF-1 (50 ng?ml) for 5 or 30 min in the presence or absence
of wortmannin (500 nM). Cells were briefly rinsed with ice-cold
PBS and lysed in 100°C 2? Nupage LDS sample loading buffer
(Invitrogen). Proteins were separated by Nupage Bis-Tris elec-
trophoresis (Invitrogen), transferred onto poly(vinylidene diflu-
oride) membranes, and analyzed by Western blotting techniques
using anti-Akt, anti-phospho-Akt (Ser-473) (Cell Signaling
Technologies), and anti-HA (Covance) antibodies per manufac-
Western Analysis. Western analysis of nuclear extracts was per-
formed with standard techniques. Proteins (30 ?g per lane) were
separated by using Nupage Bis-Tris electrophoresis (Invitrogen)
and transferred onto poly(vinylidene difluoride) membranes.
Yeast Strains. Wild-type (strain BY4741) and all yeast mutants,
with the exception of plc1?ipmk? double mutant yeast, were
obtained from Research Genetics?Invitrogen (15). plc1?ipmk?
double mutant yeast were generated by using standard homol-
ogous recombination techniques (16). The entire PLC1 gene in
the ipmk? strain was replaced by LEU2 generated by PCR
amplification of the marker gene from the PRS405 plasmid.
Yeast Growth Conditions, Transformations, and Drug Treatments.
Yeast were grown in standard yeast extract?peptone?dextrose
(YPD) or synthetic medium supplemented with a complete or
appropriate amino acid mixture of the Synthetic Complete
supplement purchased from Qbiogene. Yeast transformations
were performed by using the Alkali-Cation Yeast Transforma-
tion kit (Qbiogene). Where indicated, heterologous expression
review see ref. 3). Also depicted is the lipid inositol PI3K activity being
described. Additional activities not shown include the phosphorylation of
Ins(4,5)P2 to Ins(1,4,5)P3 and the phosphorylation of Ins(1,4,5)P3 first to
Ins(1,4,5,6)P4 and subsequently to Ins(1,3,4,5,6)P5. More recently, human
IPMK was shown to be capable of phosphorylating Ins(1,3,4,6)P4 to
www.pnas.org?cgi?doi?10.1073?pnas.0506184102 Resnick et al.
of proteins was achieved by using either pRS415 or p426-GPD
yeast expression vectors (17).
Northern Analysis. For Northern analysis, yeast were grown in
standard yeast extract?peptone?dextrose (YPD) or in synthetic
defined (SD) drop-out medium (Qbiogene). Yeast was grown to
phenol-chloroform techniques. Probes to ARG8, GAPDH, and
ACT1 were made by random priming from a PCR product of
each full-length gene.
Results and Discussion
To explore the lipid kinase activities of IPK family members, we
incubated mammalian and yeast IPMKs and IP6Ks with bovine
brain lipid extracts or PI(4,5)P2in the presence of [?-32P]ATP
(Fig. 2 A–C). No lipid kinase activity is detectable with any of the
IP6Ks tested (data not shown). In contrast, with three distinct
bovine brain lipid extracts as well as with purified PI(4,5)P2, both
yeast and mammalian IPMKs generate a lipid inositol product
comigrating on TLC with PI(3,4,5)P3produced by recombinant
p110?, a PI3K catalytic subunit. Whereas yeast and mammalian
IPMKs form only a single product, PI3K also forms PI(3,4)P2
and PI (3)P in addition to PI(3,4,5)P3when incubated with lipid
extracts, consistent with previous reports (9). Thus, IPMK is
more selective than class I PI3Ks in its lipid catalytic activity.
To delineate the specificity of IPMK, we incubated it with
[?-32P]ATP and a variety of synthetic lipid inositols (Fig. 2D).
Catalytic activity is evident only with PI(4,5)P2,further empha-
sizing the selectivity of IPMK. Yeast and rat IPMK display
similar kinetic parameters to those observed for canonical PI3Ks
(18) with apparent Kmvalues for PI(4,5)P2of 30 ?M and 6 ?M,
respectively (Fig. 7, which is published as supporting information
on the PNAS web site). Maximal specific activities for yeast and
rat IPMKs are ?0.5 ?mol?min?mg and ?0.7 ?mol?min?mg,
Human PI3K p110? (A), rat IPMK (B), and yeast IPMK (C) were incubated with
PI(4,5)P2and [?-32P]ATP in the presence of increasing concentrations of wort-
mannin. Reaction products were separated by TLC and analyzed by autora-
Yeast and mammalian IPMKs are wortmannin-insensitive PI3Ks.
inositols. Yeast IPMK (A), rat IPMK (B), or recombinant human PI3K p110? (C) were incubated in the presence of [?-32P]ATP with type III, type I, or
phosphoinositide-enriched type I bovine Folch extracts as well as purified PI(4,5)P2. (D) IPMK displays substrate specificity producing phosphorylated reaction
products only in the presence of PI(4,5)P2. Lipid reaction products were separated by TLC and analyzed by autoradiography. (E) Definitive identification of IPMK
lipid kinase reaction products was obtained by HPLC analysis and comparisons of deacylated lipids (glycerophosphoinositides) isolated from in vitro reactions
with yeast IPMK, rat IPMK, and PI3K p110? using PI(4,5)P2as substrate. More polar peaks eluting after PI(3,4,5)P3are likely artifacts of chemical deacylation and
appear to comigrate with IP4.
Resnick et al.
September 6, 2005 ?
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respectively (Fig. 7). Competition assays in which yeast and rat
IPMK lipid kinase reactions are performed in the presence of
known soluble inositol substrates suggest that PI(4,5)P2success-
fully competes with either IP3or inositol 1,3,4,6-tetrakisphos-
(19) (Fig. 8, which is published as supporting information on the
PNAS web site).
To obtain definitive evidence for the catalytic step, we mon-
itored the formation of PI(3,4,5)P3 by HPLC, analyzing the
purified, deacylated reaction products. With both yeast and
mammalian IPMKs, we observe almost exclusive formation of
PI(3,4,5)P3 whose migratory properties are identical to those
observed for PI3K reaction products (Fig. 2E). Further confir-
mation of the reaction products as the physiologically relevant
PI(3,4,5)P3isomer was obtained by incubation of the reaction
and TENsin homologue deleted on chromosome 10), a
PI(3,4,5)P3-3 phosphatase, or SHIP-2 (Src Homology 2 domain-
containing Inositol Phosphatase), a PI(3,4,5)P3-5 phosphatase.
PTEN phosphatase treatment of the reaction products results in
the loss of the radiolabeled phosphate consistent with PTEN?s
3-phosphatase activity and the presumed D-3 position labeling
by [?-32P]ATP. SHIP-2 phosphatase treatment results in a
radiolabeled product comigrating with a PI(3,4)P2 standard
fitting with its 5-phosphatase specificity (Fig. 9, which is pub-
lished as supporting information on the PNAS web site).
A hallmark of PI3K family members is their potent inhibition
by wortmannin, a naturally occurring fungal metabolite that
covalently modifies and inactivates PI3K (8). Wortmannin in-
hibits p110? PI3K at concentrations as low as 5 nM, but fails to
influence mammalian or yeast IPMK lipid kinase activity at
concentrations as high as 10 ?M (Fig. 3). Similarly, IPMK is
unaffected by LY294002, a second selective PI3K inhibitor, at
concentrations as high as 500 ?M (data not shown).
To ascertain whether IPMK possesses lipid kinase activity in
intact cells, we transfected Cos-7 cells with HA-IPMK (Fig. 4).
As described for yeast (20) and human (21) IPMK, we detected
HA-IPMK almost exclusively in nuclei. Using PI(3,4,5)P3-
specific antibodies, we observed pronounced nuclear staining for
PI(3,4,5)P3 in IPMK-transfected cells, whereas in mock-
transfected cells, PI(3,4,5)P3is almost entirely cytosolic (Fig. 4
A–F). Isolated, intact nuclei from transfected cells also display
wortmannin-insensitive PI3K activity in vitro when incubated
with [?-32P]ATP and PI(4,5)P2(Fig. 10, which is published as
supporting information on the PNAS web site). Biochemical
analysis further confirms that IPMK forms PI(3,4,5)P3in intact
cells. HPLC analysis of [3H]inositol-labeled cells transfected
fected with HA-IPMK (A–C) or empty vector (D–F) were fixed and stained via
E, red) antibodies. Merged images of cells transfected with HA-IPMK (C) or
empty vector (F) reveal an increase in nuclear PI(3,4,5)P3staining in HA-IPMK-
transfected cells that colocalize with nuclear HA-IPMK (overlap shown in
yellow). Images were acquired by confocal microscopy. Biochemical identifi-
cation of increases in PI(3,4,5)P3 in HA-IPMK-transfected cells was accom-
plished by HPLC analysis (G) of glycerophosphoinositides prepared from
[3H]inositol-labeled cells overexpressing HA-IPMK or GFP.
In vivo production of nuclear PI(3,4,5)P3by IPMK. Cos-7 cells trans-
recognizes a single band of the predicted molecular size. Immunolabeling of
E17–18 rat embryo primary cortical (B) and hippocampal (C) cultures was
performed by using anti-IPMK (green) and either anti-?III-tubulin or anti-
neurofilament (cytoplasmic neuronal markers) (red) antibodies. (D) Similar
punctuate, nuclear localization of IPMK (green) is evident in adult rat hepa-
tocytes. Hoechst staining (not shown) was used to verify nuclear localization.
Images were acquired by confocal microscopy. (E) Immunoprecipitation of
endogenous IPMK from nuclear lysates of adult rat liver or cortical cultures
reveals wortmannin-resistant PI3K activity. Immunoprecipitates were incu-
bated in the presence of [?-32P]ATP and PI(4,5)P2; reaction products were
separated by TLC and analyzed by autoradiography. Control reactions per-
formed with PI3K p85 immunoprecipitates of whole-cell, cortical culture
lysates display wortmannin-sensitive PI3K activities (E). Rabbit IgG fails to
immunoprecipitate any PI3K activity.
Endogenous IPMK localizes to the nucleus with a punctuate dispo-
www.pnas.org?cgi?doi?10.1073?pnas.0506184102Resnick et al.
with HA-IPMK reveals a significant increase in PI(3,4,5)P3
levels when compared to mock-transfected cells (Fig. 4G).
To assess the disposition and lipid kinase activity of endoge-
nous IPMK, we used antibodies raised against rat IPMK. West-
liver lysates reveals a single band of ?44 kDa, corresponding to
the predicted molecular size of IPMK (Fig. 5A). In cerebral
cortical, hippocampal, and hepatocyte cultures, IPMK immuno-
reactivity is predominantly nuclear and occurs in a punctuate
fashion (Fig. 5 B–D).
We examined the lipid kinase activity of endogenous IPMK in
rat liver and cerebral cortical cultures after immunoprecipitation
of IPMK (Fig. 5E). In immunoprecipitates from isolated nuclear
lysates, we observed formation of PI(3,4,5)P3, which is unaf-
fected by 500 nM wortmannin. The pattern of lipid kinase
activity observed is essentially the same as that observed with
immunoprecipitated PI3K, although in the latter case enzyme
activity is abolished by wortmannin.
To ascertain whether the nuclear PI(3,4,5)P3 produced by
IPMK influences the canonical PI3K signaling cascade, we
monitored levels of phospho-Akt, reflecting activation of the
PI3K pathway (Fig. 11, which is published as supporting infor-
mation on the PNAS web site) (8). Phospho-Akt levels are
unaffected by overexpression of wild-type or kinase-dead IPMK
under a variety of serum starvation or stimulation paradigms,
consistent with IPMK’s lack of involvement in the canonical
PI3K signaling pathway.
Although a physiologic function for IPMK has not been
delineated in mammalian tissues, reports (22, 23) have suggested
that yeast IPMK (also known as ArgRIII, Arg82, or Ipk2)
functions as a regulatory component of the arginine-sensitive
ArgR-Mcm1 transcriptional complex required for the suppres-
sion or induction of several gene products mediating arginine
catabolism and synthesis. We focused on ARG8 (acetylornithine
aminotransferase), whose mRNA levels are markedly aug-
mented in ipmk? yeast. Using Northern analysis, we confirm the
pronounced up-regulation of ARG8 mRNA in ipmk? yeast (Fig.
6 A and B). We show that this up-regulation is caused by the loss
of IPMK, as transformation of the deficient yeast with yeast
IPMK abolishes the up-regulation of ARG8 mRNA. Moreover,
for the production of IP6and IP7, does not affect ARG8 mRNA
The kinase activity of IPMK is required for the regulation of
dead IPMK (K133A) fails to rescue up-regulated ARG8 mRNA
levels (Fig. 6A). We wondered whether regulation of ARG8
involves the inositol kinase or the lipid kinase activities of IPMK.
Because yeast possess only one PLC isoform, phenotypic com-
parisons of plc1? yeast with yeast lacking downstream inositol
polyphosphosphate kinases has clarified functional roles for
inositol polyphosphate metabolism (4, 24–26). Despite having
no detectable inositol polyphosphates (Fig. 6C), plc1? yeast
display wild-type levels of ARG8 mRNA (Fig. 5B), suggesting
that inositol polyphosphate synthesis is not necessary for ARG8
regulation. However, ipmk? yeast possess aberrantly high levels
of IP2 and IP3 (Fig. 6C), which may function as negative
regulators of ARG8 repression. To exclude a role for water-
soluble inositol polyphosphates in ARG8 disregulation in ipmk?
yeast, we generated plc1?ipmk? double mutant yeast that, like
plc1? yeast, display a lack of higher inositol polyphosphates (Fig.
6C). Like ipmk? yeast, plc1? ipmk? yeast display elevated ARG8
mRNA levels (Fig. 6B) that can only be rescued with enzymat-
ically active yeast IPMK (data not shown). Thus, the ARG8
transcription regulatory activity of IPMK appears to reflect its
lipid inositol kinase rather than its water-soluble inositol kinase
dependent transcriptional regulation by IPMK. (A) Wild-type, ipk1?, and
ip6k? yeast transformed with empty vector (p415, LEU2) show low levels of
ARG8 expression when grown in synthetic drop-out medium. In contrast,
ipmk? yeast show robust derepression of ARG8. The ARG8 phenotype of
kinase-dead (K133?A) IPMK. A deletion of an aspartate-rich region of IPMK
(amino acids 285–300) partially impairs the ability of IPMK to rescue the ARG8
phenotype of ipmk? yeast. plc1? yeast, which lack detectable soluble inositol
polyphosphates (C), display wild-type, repressed levels of ARG8 (B), whereas
plc1?ipmk? yeast display elevated ARG8 mRNA levels (B) despite possessing a
nearly identical absence of inositol polyphosphates (C).
Northern analyses of ARG8 mRNA levels in yeast reveal lipid-kinase-
Resnick et al.
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vol. 102 ?
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Previous reports have identified a necessary role for IPMK in Download full-text
stabilizing the ArgR-Mcm1 transcriptional complex (composed
of yeast IPMK, Arg80, Arg81, and Mcm1) via direct interactions
with Mcm1 and Arg80 (20). With the exception of the catalytic
domains, rat IPMK shares little homology to yeast IPMK and
lacks conserved regulatory domains necessary for mediating
IPMK’s protein–protein interactions in yeast (20). Consistent
IPMK in mediating ARG8 regulation, overexpression of rat
IPMK in ipmk? or plc1?ipmk? yeast fails to rescue the ARG8
phenotype (data not shown).
Our findings reveal IPMK to be a robust evolutionarily
conserved inositol lipid kinase with PI3K activity. IPMK dem-
onstrates greater substrate specificity than class I PI3Ks, as
IPMK phosphorylates only PI(4,5)P2. Also, whereas class I
PI3Ks are predominantly cytosolic and are potently inhibited by
wortmannin, IPMK is almost exclusively nuclear and is unaf-
fected by wortmannin or other PI3K selective inhibitors.
Very recently (27, 28), the first crystal structures were solved
for an IPK family member, the exclusively metazoan IP3-3
of catalysis and constraints on substrate specificity for the entire
IPK family. Structure database comparisons revealed that IP3-3
kinases possess significant structural similarity to type II? phos-
phatidylinositol phosphate kinases, suggesting a direct relation-
ship between the evolutionary development of lipid inositol
kinase activities and soluble inositol kinase activities. However,
structural constraints unique to the evolutionarily recent IP3-3
kinases preclude IP3-3 kinases from functioning as lipid kinases
(27, 28). These constraints are absent in yeast and mammalian
IPMKs, allowing for both lipid and soluble inositol kinase
Although exact functions of IPMK-mediated nuclear
PI(3,4,5)P3 production in mammalian tissues are unclear, in
yeast, IPMK PI3K activity appears to regulate transcription.
Functional roles for soluble inositol polyphosphate kinase ac-
tivity in transcriptional regulation and chromatin remodeling
have been proposed (4, 25), and kinase-independent transcrip-
tional regulation has also been suggested (24). Our identification
the analysis of transcriptional regulation permitting clarification
of previously conflicting interpretations.
Most studies of signaling pathways regulated by PI3K have
focused on tyrosine kinase-linked receptors and their associated
cytosolic signaling cascades. However, selective nuclear lipid
inositol pathways have been extensively described (29, 30).
Critical elements of the inositol lipid kinase pathways occur in
and the lipid inositol phosphatases, PTEN and SHIP, that
dephosphorylate PI(3,4,5)P3 (1, 30). Nuclear, transcriptional
regulatory proteins that act as receptors for lipid inositols,
including PI(3,4,5)P3, have also been identified (31). Whereas
cytosolic lipids often comprise classic lipid bilayers, nuclear
inositol lipids are detergent-resistant and not associated with
nuclear membranes (29). Thus, alternative techniques may be
necessary to elucidate inositol lipid dynamics and regulation in
nuclei. The absence in yeast of receptor-tyrosine kinase path-
ways and associated class I PI3Ks, as well as the inability to
biochemically detect PI(3,4,5)P3 in yeast extracts had implied
that PI(3,4,5)P3 signaling is relegated to higher organisms,
although in animal tissues, PI(3,4,5)P3is thought to comprise
?0.005% of total inositol lipids (32). However, the recent
discovery of PI(3,4,5)P3in fission yeast lacking a PTEN homo-
logue (33) confirms the existence of more primitive biosynthetic
pathways for the lipid inositol. Our demonstration that IPMK is
a robust physiologic nuclear PI3K that regulates transcription
implies functional importance for PI(3,4,5)P3 in the nuclei of
both mammals and yeast.
We sincerely thank Lynda Hester for her technical expertise and
assistance. This work was supported by United States Public Health
Service Grant DA-000266 (to S.H.S.).
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