Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase.

Abstract

Extracellular signaling molecules bound to cell surface receptors can regulate nuclear function with consequences for cell proliferation, differentiation, and function. To regulate nuclear function, signals must be transduced across the nuclear envelope to propagate the signal from the cytoplasm to the nucleus. Therefore, many signaling responses induce the nuclear translocation of transcription factors, kinases, and others. By using inducible translocation trap, a reporter gene-based system to detect inducible nuclear translocation, we found that the M2 isoform of pyruvate kinase, a key enzyme in glycolysis, translocates into the nucleus by interleukin-3, but not by epidermal growth factor, stimulation. The C domain of the M2 isoform of pyruvate kinase was sufficient for interleukin-3-induced nuclear translocation. Interleukin-3-induced nuclear translocation of the M2 isoform of pyruvate kinase was dependent on the activation of Jak2. Overexpression of the M2 isoform of pyruvate kinase protein fused with a nuclear localization signal enhanced cell proliferation in the absence of interleukin-3, suggesting that the nuclear pyruvate kinase plays an important role in cell proliferation.

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Regulation of Cell Proliferation by Interleukin-3-induced
Nuclear Translocation of Pyruvate Kinase*□
Receivedforpublication,January4,2007,andinrevisedform,April17,2007 Published,JBCPapersinPress,April19,2007,DOI10.1074/jbc.M700094200
Akemi Hoshino, John A. Hirst, and Hodaka Fujii1
FromtheDepartmentofPathology,NewYorkUniversitySchoolofMedicine,NewYork,NewYork10016
S
Extracellularsignalingmoleculesboundtocellsurfacerecep-
tors can regulate nuclear function with consequences for cell
proliferation, differentiation, and function. To regulate nuclear
function, signals must be transduced across the nuclear enve-
lope to propagate the signal from the cytoplasm to the nucleus.
Therefore, many signaling responses induce the nuclear trans-
location of transcription factors, kinases, and others. By using
inducible translocation trap, a reporter gene-based system to
detect inducible nuclear translocation, we found that the M2
isoform of pyruvate kinase, a key enzyme in glycolysis, translo-
cates into the nucleus by interleukin-3, but not by epidermal
growthfactor,stimulation.The C domain of the M2 isoform of
pyruvate kinase was sufficient for interleukin-3-induced
nuclear translocation. Interleukin-3-induced nuclear trans-
location of the M2 isoform of pyruvate kinase was dependent
on the activation of Jak2. Overexpression of the M2 isoform
of pyruvate kinase protein fused with a nuclear localization
signal enhanced cell proliferation in the absence of interleu-
kin-3, suggesting that the nuclear pyruvate kinase plays an
important role in cell proliferation.
Interleukin-3(IL-3)2stimulationinducesactivationofJak2
(1), and activated Jak2 phosphorylates and activates Stat5a/b
(2–4). Accumulating evidence suggests critical roles of Stat5
activation in IL-3 signaling (5–7). On the other hand, Stat5-
independent induction of target genes has also been
reported (7), suggesting that molecules other than Stat5a/b
may translocate into the nucleus by IL-3 stimulation. Here,
we showed IL-3-induced nuclear translocation of the M2
isoform of pyruvate kinase (M2-PK) and examined its signif-
icance in cell proliferation.
EXPERIMENTAL PROCEDURES
Cell Lines—BB13 was derived from an IL-3-dependent
hematopoietic cell line, Ba/F3 (8), and ectopically expresses
human epidermal growth factor (EGF) receptor and an anti-
apoptotic protein, Bcl-2 (9). BB13 proliferates in response to
EGFevenintheabsenceofIL-3.Bcl-2expressionisrequiredfor
EGF-induced proliferation. In the absence of EGF and IL-3,
BB13diesbyapoptosis,butcelldeathismarkedlydelayedcom-
pared with its parental cell line, which does not overexpress
Bcl-2. LexA-d1EGFP (10) gene was transfected into BB13 to
establish the BBLG cell line. LexA-d1EGFP reporter gene con-
tains 8 ? LexA binding elements, interferon ? minimal pro-
moter, and destabilized enhanced green fluorescent protein
(GFP) (d1EGFP).
Plasmid Construction—For construction of a retroviral vec-
tor expressing GFP fused with the carboxyl (C)-terminal of the
full-length M2-PK (M2-PK-GFP), the coding sequence of
mouse M2-PK was amplified by PCR from the Ba/F3 cDNA
library and ligated with GFP gene from pEGFP-N3 (Clontech)
and pMX-neo vector. For construction of a retroviral vector
expressing hemagglutinin (HA) epitope-tagged M2-PK fused
withthenuclearlocalizationsignal(NLS)fromSV40T-antigen
at its N terminus (HA-NLS-M2-PK), the coding sequence of
M2-PK was amplified by PCR and ligated with a sequence
encoding SV40 T-antigen NLS cleaved from pLGV-NLS-?-ga-
lactosidase (10) and pEF-HA (11). The HA-NLS-M2-PK
sequence was cleaved from the resultant plasmid and ligated
with pMXs-IG (12) (HA-NLS-M2-PK/pMXs-IG). For con-
struction of a retroviral vector encoding HA-tagged M2-PK
(HA-M2-PK/pMXs-IG), the coding sequence of M2-PK was
amplified by PCR and ligated with HA sequence from pEF-HA
into pMXs-IG.
Immunoblot Analysis—Preparation of nuclear extracts/
whole cell lysates and immunoblot analyses were performed as
described(10,13).Theantibodies(Abs)usedinthisstudywere
anti-PK (Abcam and Serotec), anti-Stat5, anti-Sp1, anti-Rho-
GDI (Santa Cruz Biotechnology), anti-GFP (Clontech), and
anti-HA (Roche Applied Science). For inhibition of Jak2,
AG-490 (BIOSOURCE) (50 ?M) was added to growth factor-
starved cells 30 min before IL-3 stimulation.
Preparation of Nuclei for Flow Cytometric Analysis—Cells
were washed with ice-cold phosphate-buffered saline, sus-
pended in 400 ?l of A buffer (10 mM Hepes-KOH, pH 7.9, 1.5
mM MgCl2, 10 mM dithiothreitol, protease inhibitor mixture
(Complete, Mini, EDTA-free; Roche Applied Science), and
0.3% Nonidet P-40), and vortexed for 30 s. Nuclei were spun
down at 5,000 rpm for 1 min at room temperature and sus-
pended in 4% paraformaldehyde to be fixed for 10 min at room
temperature. Fixed nuclei were washed with flow cytometry
buffer (phosphate-buffered saline containing 0.5% bovine
* ThisworkwassupportedbyNationalInstitutesofHealthGrantAI059315(to
H. F.). The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-
cate this fact.
□
STheon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental Figs. S1 and S2.
1To whom correspondence should be addressed: Dept. of Pathology, New
YorkUniversitySchoolofMedicine,550FirstAve.,NewYork,NY10016.Tel.:
212-263-8514; Fax: 212-263-8179; E-mail: hodaka@med.nyu.edu.
2Theabbreviationsusedare:IL,interleukin;PK,pyruvatekinase;EGF,epider-
mal growth factor; Stat, signal transducer and activator of transcription;
GFP, green fluorescent protein; EGFP, enhanced GFP; HA, hemagglutinin;
NLS, nuclear localization signal; Ab, antibody.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 24, pp. 17706–17711, June 15, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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www.jbc.org
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Supplemental Material can be found at:

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serum albumin) and suspended in flow cytometry buffer for
flow cytometric analysis.
Fluorescent Microscopy—Ba/F3 expressing M2-PK-GFP
weregrowthfactor-starvedandmock-stimulatedorstimulated
with IL-3 for 30 min. Cells were fixed with paraformaldehyde,
counterstainedwith4?,6-diamidino-2-phenylindoletoindicate
the positions of the nuclei, and observed with a deconvolution
microscope.
NorthernBlotAnalysis—Cellsweregrowthfactor-starvedfor
5 h and mock-stimulated or stimulated with EGF (10 ng/ml) or
IL-3 (1 ng/ml) for the indicated time intervals. Total RNA (5
?g) were analyzed by Northern blot analysis with cis (14) and
oncostatin M (15) probes as described previously (16, 17).
RESULTS
Isolation of M2-PK cDNA in Screening of IL-3-induced
Nuclear Translocating Molecules—By using the inducible
translocation trap system (10), we attempted to isolate cDNAs
that encode proteins translocating into the nucleus by IL-3
stimulation. The inducible translocation trap system is based
on expression of a fusion protein consisting of the LexA DNA-
binding domain, the Gal4 transactivation domain, and the test
protein encoded by cDNA subcloned downstream of the Gal4
transactivation domain (10). The fusion molecule is expressed
incellscontainingGFPreportergenewithmultipleLexAbind-
ing sites in its promoter (LexA-d1EGFP). Following nuclear
translocation of the fusion protein by ligand stimulation, the
LexA DNA-binding domain targets the fusion protein to the
LexA operator sites of the reporter gene and then the Gal4
transactivation domain activates the expression of GFP. Thus,
nuclear translocation of the test
protein is detected by the expres-
sion of GFP. LexA-d1EGFP gene
was transfected into BB13 (9) to
establish the BBLG cell line. BB13 is
a Ba/F3-derived cell line ectopically
expressing EGF receptor and anti-
apoptotic protein Bcl-2. Parental
Ba/F3 dies by apoptosis in the
absence of IL-3, whereas BB13 pro-
liferates even in the absence of IL-3
if EGF is present in the culture
medium (9).
Screening of the cDNA library
was performed as schematized in
Fig. 1. The Ba/F3 cDNA library (48
?g) constructed in the pLG retrovi-
ral vector to express cDNA-encoded
proteins fused with LexA DNA-
binding domain and Gal4 transac-
tivation domain (10) was trans-
fected into 1.8 ? 107of 293T cells
with amphotropic helper plasmid
to produce retrovirus particles.
Two days after transfection, 2 ?
107of BBLG were infected with
the supernatant (60 ml) of the
293T containing virus particles.
Infection efficiency was estimated to be ?7% (data not
shown). A day after infection, cells were resuspended in
medium containing EGF (10 ng/ml). Two days after infec-
tion, cells were stimulated with IL-3 (1 ng/ml) for 4 h, and
GFP (?) cells were sorted and incubated in medium contain-
ing EGF (IL-3 (?)) for 4 days for down-regulation of GFP,
and then GFP (?) cells were sorted. Three days after, cells
were restimulated with IL-3, and GFP (?) cells were sorted.
Nine rounds of GFP (?) sorting after IL-3 stimulation and
subsequent GFP (?) sorting were performed. The percent-
age of GFP (?) cells did not increase by IL-3 stimulation 2
days after infection (mock-stimulation, 1.57%; IL-3 stimula-
tion, 1.56%) (Fig. 2A). In contrast, after nine rounds of sort-
ing, IL-3 stimulation significantly increased GFP (?) cells in
the sorted population (mock-stimulation, 17.1%; IL-3 stim-
ulation, 33.6%) (Fig. 2A). Then cells were subjected to single-
cell sorting, and GFP induction by IL-3 was examined for
each clone. About 240 of more than 600 clones showed IL-3-
induced GFP expression. Representative data for clones that
showed GFP induction by IL-3 are shown in Fig. 2B. PCR
amplification using genomic DNA extracted from these
clones and viral vector primers gave rise to several different
patterns (Fig. 2C, a–d). Percentages of each pattern were
84% (a), 8% (b), 4% (c), 1% (d), and 3%, others. We concen-
trated our effort for the analysis of the cDNA inserts in the
dominant pattern (a). The fact that three bands were ampli-
fied in the pattern (a) suggested that three different cDNA
clones are integrated in the genome of this clone. To deter-
mine which cDNA insert is responsible for IL-3-induced
expression of GFP, PCR products from the pattern (a) were
FIGURE 1. Scheme of screening of cDNA library to identify proteins that translocate into the nucleus by
IL-3 stimulation.
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subcloned into pLG and transduced into BBLG, and then
GFP expression by IL-3 was analyzed. IL-3 stimulation sig-
nificantlyinducedexpressionofGFPinBBLGexpressingthe
fusion protein consisting of LG and the protein encoded by
the 1.2-kb pair (kbp) insert (Fig. 2D). In contrast, IL-3 stim-
ulation did not induce expression of GFP in BBLG express-
ing fusion proteins consisting of LG and proteins encoded by
the other inserts (1.6- and 0.8-kbp) (Fig. 2D). These data
indicate that the protein encoded by the 1.2-kbp insert of the
pattern (a) translocates into the nucleus by IL-3. Sequence
analysis revealed that the 1.2-kbp insert encodes the C-ter-
minal 139 amino acid residues of mouse M2-PK (18) (Fig.
2E). PK is a key enzyme in the glycolytic pathway, and it
catalyzes the transfer to ADP of the high energy phosphate
group of phosphoenolpyruvate to generate ATP (19, 20).
Four highly homologous isoforms, M1, M2, L, and R types,
have been reported in mammalian cells (21–23). M2-PK is
localizedmainlyinthecytoplasmandexpressedinearlyfetal
tissues as well as in most of the adult tissues (19, 20).
Nuclear Translocation of M2-PK by IL-3—To examine
whether endogenous PK translocates into the nucleus by IL-3,
Ba/F3 was growth factor-starved and stimulated with IL-3.
Nuclear extracts were subjected to immunoblot analysis with
anti-PKAb,whichdetectsM2-PK(seebelow).IL-3stimulation
FIGURE 2. Molecular cloning of M2-PK cDNA by inducible translocation trap. A, Ba/F3-derived cDNA library was transduced into BBLG cells. Transduced
cells were maintained in medium containing EGF (10 ng/ml) (right panels) or stimulated with IL-3 (1 ng/ml) (left panels) for 5 h, and then GFP expression was
examined.Upperpanels,beforesorting;lowerpanels,afternineroundsofsorting.B,IL-3-inducedup-regulationofGFPinsortedclones.Cellsweremaintained
in medium containing EGF (thin line) or stimulated with IL-3 (thick line) for 5 h. C, cDNA inserts amplified by PCR using genomic DNAs extracted from
IL-3-responsive clones and viral vector primers. Percentages of each pattern (a–d) in the total of ?240 clones are indicated. D, IL-3-induced up-regulation of
GFP in BBLG expressing a fusion protein consisting of LG and the protein encoded by the 1.2-kbp cDNA insert from the pattern (a). PCR products from the
pattern (a) subcloned into pLG were transduced into BBLG. Cells were stimulated with IL-3 for 4 h, and then GFP expression was analyzed by flow cytometry.
E, schematic view of M2-PK. The 1.2-kbp fragment encoded amino acids 393–531 of the C domain of M2-PK.
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inducedincreasesintheamountsofPKinthenucleuswithin30
min(Fig.3A,upperpanel).ThetotalexpressionlevelsofPKdid
not change by IL-3 stimulation (Fig. 3A, lower panel), suggest-
ing that the increase in the amounts of PK in the nucleus is not
causedbytheincreaseintheexpressionlevelsofPK.Incontrast
to IL-3, EGF stimulation did not increase the amounts of PK in
the nucleus (Fig. 3B).
To further confirm nuclear translocation of M2-PK by IL-3
stimulation, a retroviral vector expressing GFP fused with the
C-terminal of the full-length M2-PK (M2-PK-GFP) was trans-
duced into Ba/F3 cells. Expression of the M2-PK-GFP fusion
protein was detected by immunoblot analysis with anti-PK Ab
(Fig. 3C, left panel). GFP (?) cells were sorted, growth factor-
starved, and then stimulated with IL-3 for 30 min. IL-3 stimu-
lation induced increases in the amounts of M2-PK-GFP in the
nuclear extracts (Fig. 3C, right panel, upper). The total expres-
sion levels of M2-PK-GFP did not change by IL-3 stimulation
(lower panel), suggesting that the increase in nuclear amounts
of M2-PK-GFP reflects its nuclear
translocation. Next, Ba/F3 express-
ing M2-PK-GFP were IL-3-starved
for 5 h and stimulated with IL-3 for
the indicated time intervals. Nuclei
were purified and subjected to flow
cytometric analysis. IL-3 stimula-
tion increased the fluorescence
intensities of GFP (Fig. 3D). Fur-
thermore,subcellularlocalizationof
M2-PK-GFP was examined by fluo-
rescent microscopy. In the absence
of IL-3, M2-PK-GFP was excluded
from the nucleus (Fig. 3E). In con-
trast,M2-PK-GFPdistributedubiq-
uitously in the cytoplasm as well as
in the nucleus in IL-3-stimulated
cells (Fig. 3E). Collectively, these
analyses showed nuclear transloca-
tion of M2-PK by IL-3 stimulation.
Treatment with AG-490, an inhibi-
tor of Jak2 (24), completely inhib-
ited nuclear translocation of PK by
IL-3 stimulation (Fig. 3F), suggest-
ingthatIL-3-inducednucleartrans-
location of PK is dependent on Jak2
activation.
FunctionalRole
M2-PK in Cell Proliferation—To
examine the functional significance
of IL-3-induced nuclear transloca-
tion of M2-PK, we generated con-
structsencodingHA-taggedM2-PK
(HA-M2-PK)and
M2-PK fused with NLS from SV40
T-antigenatitsNterminus(Fig.4A,
HA-NLS-M2-PK). These constructs
wereinsertedintothepMXs-IGret-
roviralvectorcontaininganinternal
ribosome entry site-GFP sequence,
ofNuclear
HA-tagged
which permits simultaneous expression of a cloned gene and
GFP(12).Changesinthepercentagesoftransgene(?)cellscan
be easily quantified by analyzing GFP (?) cells. The retroviral
constructs were transduced into BB13 and cultured in RPMI
medium containing EGF. As expected, in the absence of IL-3,
HA-NLS-M2-PK was readily detected in the nucleus of GFP
(?) BB13 cells, while marginal amounts of HA-M2-PK were
detected in the nucleus (Fig. 4B).
Next,weexaminedtheproliferationofBB13constitutively
expressing M2-PK in the nucleus by monitoring the percent-
ages of transgene (?) (GFP (?)) cells in EGF-containing
medium. The percentages of GFP (?) cells in cell popula-
tions transduced with HA-M2-PK/pMXs-IG or pMXs-IG
decreased gradually (Fig. 4C), indicating that neither trans-
gene enhances proliferation. In contrast, the percentages of
GFP (?) cells in the cell population transduced with
HA-NLS-M2-PK/pMXs-IGincreasedmarkedlyinEGF-con-
taining medium (Fig. 4C). After 30 days of culture, the per-
FIGURE3.NucleartranslocationofPKbyIL-3.A,Ba/F3wasgrowthfactor-starvedandstimulatedwithIL-3for
the indicated time intervals. Nuclear extracts were subjected to immunoblot analysis. Expression levels of PK
weremonitoredbyimmunoblotanalysisofwholecelllysates.B,EGFdoesnotinducenucleartranslocationof
PK.BB13weregrowthfactor-starvedandstimulatedwithEGFfortheindicatedtimeintervals.Nuclearextracts
wereprepared,andimmunoblotanalysiswasperformedasdescribedabove.C,Ba/F3wastransducedwiththe
retroviral vector expressing M2-PK-GFP. Expression of M2-PK-GFP was monitored by immunoblot analysis
usinganti-PKAb(leftpanel).Cellsweregrowthfactor-starvedandstimulatedwithIL-3for30min.Immunoblot
analysis of nuclear extracts was performed with anti-GFP or anti-Stat5 Ab (right panel). D, flow cytometric
analysisofincreaseintheamountsofnuclearM2-PK-GFPbyIL-3stimulation.Ba/F3expressingM2-PK-GFPwas
stimulated with IL-3 for the indicated time intervals. Nuclei were purified and subjected to flow cytometric
analysis. Fluorescence intensities of GFP were normalized against that of the unstimulated sample. E, fluores-
centmicroscopyofM2-PK-GFP.Ba/F3expressingM2-PK-GFPwasgrowthfactor-starvedandmock-stimulated
or stimulated with IL-3 for 30 min. Cells were fixed, counter-stained with 4?,6-diamidino-2-phenylindole to
indicate the positions of the nuclei, and observed with a deconvolution microscope. F, IL-3-induced nuclear
translocation of PK depends on activation of Jak2. Ba/F3 was growth factor-starved for 5 h and mock-stimu-
latedorstimulatedwithIL-3(1ng/ml)for30min.AG-490(50?M)wasadded30minbeforestimulation.Nuclear
extracts were subjected to immunoblot analysis with anti-PK (upper).
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centage of GFP (?) cells expressing HA-NLS-M2-PK
increased 3-fold (Fig. 4D). These data indicate that nuclear
M2-PK enhances EGF-induced proliferation. We also
observed enhanced proliferation of clones expressing
HA-NLS-M2-PK (data not shown). It is noteworthy that
enhanced proliferation of cells expressing HA-NLS-M2-PK
in the presence of IL-3 was not observed (supplemental Fig.
S1), consistent with the idea that IL-3 stimulation induces
nuclear translocation of sufficient amounts of M2-PK for
maximal cell proliferation. In addition, because survival of
cells expressing HA-NLS-M2-PK
was still dependent on EGF or IL-3
(supplementalFig.S2),thenuclear
localization of M2-PK alone is not
sufficient for cell proliferation and
survival.
DISCUSSION
Here, we showed that M2-PK
translocates into the nucleus by
IL-3 but not by EGF stimulation
(Figs. 2 and 3). Because sequence
identities of isoforms of mouse
PK are high, it is likely that
other isoforms (L, R, and M1) of
PK can also translocate into the
nucleus by IL-3. It has been
recently shown that growth fac-
tors, including IL-3, influence cell
growthand survival
effects on glucose metabolism
(25). Our data show that growth
factors also modulate localization
of PK, a key enzyme in glycolysis,
suggesting a novel link between
growth factor stimulation and the
glycolytic pathway.
PK proteins consist of the N-ter-
minal domain, the A domain subdi-
vided into A1 and A2 subdomains,
the B domain, and the C domain
(Fig. 2E) (26). The ADP/ATP bind-
ing site lies in subdomain A2 near
the domain C (26). The binding site
of phosphoenolpyruvate is located
in the loops that connect subdo-
main A1 and B and strands in sub-
domain A2 (27). The active site lies
in a pocket between domains A and
B (27), where there is a high degree
of identity among the 50 PK
sequences of organisms from bacte-
ria to human (26). In contrast, the C
domain is variable among species
(26). We showed that the C domain
contains an inducible NLS(s) (Fig.
2). On-line motif analysis with the
PSORTIIprogram
through
(psort.ims.
u-tokyo.ac.jp) did not detect any classical and bipartite NLS
richinArgandLys(28)intheCdomainofPK.Inthisregard,it
is known that signaling proteins that inducibly translocate into
the nucleus in response to extracellular stimuli such as Stat
proteins do not necessarily contain Arg- or Lys-rich NLS (29).
Instead, their signal-induced conformational changes can be
recognized by nuclear importer proteins such as importin/
karyopherins. Nuclear translocation of PK may also be medi-
ated through signal-induced conformational changes but not
by classical/bipartite NLS.
FIGURE4.EnhancedEGF-mediatedcellproliferationbynuclearM2-PK.A,schematicviewofHA-M2-PK
andHA-NLS-M2-PK.B,BB13wastransducedwithconstructsinpMXs-IG,andGFP(?)cellsweresortedand
cultured in medium containing EGF. The amounts of nuclear HA-M2-PK or HA-NLS-M2-PK were examined
by immunoblot analysis with anti-HA Ab. C, increased percentages of GFP (?) (transgene (?)) cells
expressingNLS-fusedM2-PK.PercentagesofGFP(?)cellsattheindicateddaysafterEGFstimulationwere
normalizedagainstthoseofday0.D,flowcytometricprofilesofBB13transducedwithHA-M2-PK/pMXs-IG
or HA-NLS-M2-PK/pMXs-IG just after transduction (Day 1) and after culture in EGF-containing medium for
30 days (Day 30). FSC, forward scatter. E, nuclear M2-PK does not induce expression of Stat5 target genes.
Cells were growth factor-starved for 5 h and mock-stimulated or stimulated with EGF (10 ng/ml) or IL-3 (1
ng/ml) for the indicated time intervals. Total RNA (5 ?g) were analyzed by Northern blot analysis with cis
and oncostatin M (osm) probes.
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We showed that IL-3-mediated nuclear translocation of PK
is dependent on activation of Jak2 (Fig. 3F). At this stage, it is
not clear how Jak2 activation induces nuclear translocation of
PK. IL-3-induced activation of Jak2 induces activation of Stat5
as well as other signaling molecules such as Ras (30) and Akt
(31, 32), and they may be involved in the regulation of nuclear
translocation of M2-PK. Alternatively, Jak2 may directly phos-
phorylate PK to induce its nuclear translocation.
The nuclear localizing M2-PK significantly enhanced EGF-
mediated cell proliferation in the absence of IL-3 (Fig. 4). It is
notclearhownuclearM2-PKenhancescellproliferation.EGF-
stimulated cells did not induce Stat5 target genes even when
HA-NLS-M2-PK was expressed (Fig. 4E), excluding a possibil-
itythatnuclearM2-PKinducesorenhancesactivationofStat5.
M2-PKmayprovideATPfornuclearfunctions,includingtran-
scription. Alternatively, PK may function as a signaling mole-
cule to modulate nuclear function. Further studies will be
required to elucidate how nuclear M2-PK enhances cell
proliferation.
Acknowledgments—We thank Drs. T. Kitamura and A. Yoshimura
forpMXs-IGvectorandcisandoncostatinMprobes,respectively.We
also thank S. Matsumura, E. Kono, and K. Kondo for technical
assistance.
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IL-3-inducedNuclearTranslocationofPyruvateKinase
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