J. Biol. Chem.
Song, Ping Miao, Li Zhao, Xiaoping Zhao and
Fajun Yang, Lei Xie, Jianjun Liu, Shaoli
Zhenhai Yu, Liangqian Huang, Teng Zhang,
Glycolysis in Cancer Cells
PIM2 phosphorylates PKM2 and promotes
published online October 18, 2013
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PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells*
Zhenhai Yu1, Xiaoping Zhao2, Liangqian Huang3, Teng Zhang2, Fajun Yang4, Lei Xie2, Shaoli Song2,
Ping Miao2, Li Zhao2, Xiaoguang Sun2, Jianjun Liu2# and Gang Huang1,2,3#
1 School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China.
2 Department of Nuclear Medicine, Renji Hospital, Shanghai Jiao Tong University, School of Medicine,
Shanghai 200127, China.
3 Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
4 Department of Medicine and Developmental & Molecular Biology, Albert Einstein College of Medicine, New
York, NY 10461, USA.
*Running title: PIM2 phosphorylates PKM2
#To whom correspondence should be addressed: Gang Huang, School of Biomedical Engineering, Shanghai Jiao
Tong University, Shanghai 200030, PR China; Institute of Health Sciences, Shanghai Jiao Tong University
School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of
Sciences (CAS), Shanghai 200025, China; Department of Nuclear Medicine, Renji Hospital Shanghai Jiao Tong
University School of Medicine, Shanghai 200127, China, Tel: +86-21-63867812,63853125. Fax: +86-21-
63842916. E-mail: firstname.lastname@example.org
Xiaoping Zhao, Department of Nuclear Medicine, Renji Hospital Shanghai Jiao Tong University School of
Medicine, Shanghai 200127, China, Tel: +86-21-68383962. Fax: +86-21-68383962. E-mail: email@example.com
Keywords: Cancer; Cell proliferation; Glycolysis; phosphorylation; Pyruvate kinase
Background: The serine/threonine protein kinase
PIM2 regulates glycolysis, but the mechanism is not
Results: PIM2 interacts with PKM2 and
phosphorylates PKM2 on the Thr454 residue.
Conclusion: This phosphorylation of PKM2 increases
glycolysis and proliferation in cancer cells.
Significance: PIM2-dependent phosphorylation of
PKM2 is critical for regulating the Warburg effect in
cancer, highlighting PIM2 as a potential therapeutic
Pyruvate kinase M2 (PKM2) is a key
player in the Warburg effect of cancer cells.
However, the mechanisms of regulating PKM2 are
not fully elucidated. Here, we identified the
serine/threonine protein kinase PIM2, a known
oncogene, as a novel binding partner of PKM2.
The interaction between PIM2 and PKM2 was
confirmed by multiple biochemical approaches in
vitro and in cultured cells. Importantly, we found
that PIM2 could directly phosphorylate PKM2 on
the Thr454 residue, resulting in an increase of
PKM2 protein levels. Compared to wild-type,
PKM2 with the phosphorylation-defective
mutation displayed a reduced effect on glycolysis,
co-activating HIF-1α and -catenin, and cell
proliferation, while enhanced
respiration of cancer cells. These findings
phosphorylation of PKM2 is critical for regulating
the Warburg effect in cancer, highlighting PIM2 as
a potential therapeutic target.
Tumor cell proliferation proceeds only when
sufficient energy and building blocks are available.
Compared to normal tissues, most tumors exhibit a
significant increase of glucose utilization, namely the
Warburg effect (1). Such characteristic of increased
glucose uptake, which accompanies the aerobic
glycolysis, has been exploited for diagnosis of cancer
using 18F-deoxyglucose position emission tomography
(FDG-PET) (2). Due to the changes of glycolytic
enzymes, tumor cells shift glucose metabolism from
oxidative phosphorylation to glycolysis even in the
presence of oxygen. To date, pyruvate kinase (PK) is
considered as a key regulator of the Warburg effect (3).
PK catalyzes the
concomitant formation of ATP, which is a rate-limiting
step in glycolysis. There are four isoenzymes of PK, L,
R, M1 and M2, which are encoded by two separate
genes. The L and R isoforms of PK, which are
http://www.jbc.org/cgi/doi/10.1074/jbc.M113.508226The latest version is at
JBC Papers in Press. Published on October 18, 2013 as Manuscript M113.508226
Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
expressed exclusively in the liver and red blood cells,
respectively, are originated from the PKL gene by
alternative splicing (4). PKM1 and PKM2 isoforms
are alternative-splicing products of the PKM gene
(exon 9 for PKM1 and exon 10 for PKM2) (5).
During embryogenesis, PKM2 is progressively
replaced by PKM1. Conversely, during tumorigenesis,
the L-PK or PKM1 isoenzymes are down regulated
and PKM2 is re-expressed, suggesting unique roles of
PKM2 in cancer cells. Since PKM2 has a lower
enzymatic activity as compared to PKM1, it will
channel more glycolytic intermediates into building
blocks, such as nucleic acids, amino acids and lipids,
to support cancer cell proliferation. The enzymatic
activity of PKM2 is under the control of metabolic
intermediates, oncogenes and growth factors (6).
Growing evidence indicates that oncogenes reprogram
glycolysis impacting tumor aggressive phenotype via
regulating PKM2 (7). In addition to its direct roles in
glycolysis, recent studies have also demonstrated that
PKM2 can function as a transcriptional co-activator or
a protein kinase to promote gene transcription and
Transcription regulation appears not to be the
primary mechanism of regulating PKM2. Throughout
mitosis, PKM2 mRNA and activity are declined
whereas the protein levels continue to increase (12).
The decrease of PKM2 activity is due to
posttranslational modifications (13). It has been
shown that acetylation of PKM2 at K305 promotes its
degradation via chaperone-mediated autophagy (14).
proteins (23). All three PIM kinases can
phosphorylate Thr-157 and Thr-198 of p27kip1,
promoting its binding to the 14-3-3 proteins, resulting
in nuclear exclusion and degradation (24). PIM1 can
phosphorylate the intracellular domain of CXCR4 at
Ser-339, a site critical for CXCR4 recycling (25).
PIM2 has been reported to phosphorylate the
ribosomal protein 4E-BP1, causing its dissociation
from Eif-4e, which impacts protein synthesis (26).
Therefore, inhibiting PIM kinases may lead to
apoptosis, cell cycle arrest and senescence. For that
reason, PIM kinase inhibitors have been actively
developed for cancer treatment (27).
Here, we identify PIM2 as a novel binding
partner of PKM2 from a yeast two-hybrid screen. We
show that PIM2 critically regulates multiple aspects of
PKM2 functions through direct phosphorylation. Thus,
our results provide a new insight into the regulation of
PKM2 and its contribution to the Warburg effect in
Interestingly, phosphorylation at tyrosine or serine
residues has been implicated in regulating PKM2. In
pp60v-src kinase transformed cells, increased tyrosine
phosphorylation of PKM2 correlates with its
inactivation (15,16). In addition, fibroblast growth
factor receptor 1 (FGFR1) phosphorylates PKM2 on
Tyr-105, which inhibits the formation of active,
tetrameric PKM2 by disrupting binding of PKM2
cofactor fructose-1, 6-biophosphate (17). Protein-
tyrosine phosphatase 1B (PTP1B) reverses this
phosphorylation (18). A-Raf can bind to and
phosphorylate PKM2 on serine residues, inducing a
transition of dimeric to tetrameric active form of
PKM2 (19). Although it is not fully clear, PKC is
believed to regulate PKM2 protein stability via
phosphorylation (20). Moreover, ERK1/2 has been
shown to phosphorylate PKM2 on Ser-37 and
promote its nuclear translocation, which is important
to tumor growth (12).
Proviral insertion in murine lymphomas (PIM)
protein kinases are highly conserved oncogenic
serine/threonine kinases and have three isoforms:
PIM1, PIM2 and PIM3 (21). It has been reported that
PIM kinases are aberrantly expressed in multiple
types of cancer (22). PIM kinases are responsible for
cell cycle regulation, anti-apoptotic activity and other
malignant phenotypes of cancer (23). PIM kinases
mediate their oncogenic
phosphorylating a wide range of cellular
Materials - Rabbit ant-PIM2 antibody was purchased
from GeneTex, rabbit anti-PKM2 antibody from
Abcam, rabbit anti-phospho-Serine antibody from
Invitrogene, rabbit anti-phospho-Threonine antibody
from Cell Signaling, mouse anti-HA, -Flag or β-actin
antibodies from Sigma, and rabbit or mouse IgG from
Santa Cruz Biotechnology. Goat anti-Mouse or Rabbit
second antibodies were purchased from LI-COR
Biosciences. The plasmids used in this study were
generated by subcloning the indicated human cDNA
fragments into expression vectors. All plasmids were
verified by DNA sequencing. The sequences for
siRNA and PCR primers are listed in Table S2.
Yeast Two-hybrid Screen - The C-terminal portion
(aa354–531) of human PKM2 was subcloned into the
yeast expression vector pGBKT7 (Clontech) in frame
with the Gal4DNA binding domain. This bait was
used to screen human cDNA libraries (Human Kidney
Matchmaker® cDNA Library pACT2 Catalog No.
638816, Human Liver Matchmaker® cDNA Library
pACT2 Catalog No. 638802 and Mate & Plate™
Library-Human Brain (Normalized) Catalog No.
630486) (Clontech). The yeast two-hybrid screens
were performed according to the manufacturer’s
Cell Culture and Transient Transfection - All cell lines
were cultured in Dulbecco’s Modified Eagle Medium
(GIBCO) supplemented with 10% fetal bovine serum
(GIBCO), 100mg/ml penicillin, and 100mg/ml
streptomycin sulfate (GIBCO) at 37C and 5% CO2.
Hypoxic treatment was performed in a specially
designed hypoxia incubator (Thermo Electron, Forma,
MA, USA) with 1% O2, 5% CO2 and 94% N2.
Lipofectamine 2000 (Invitrogen) was used in transient
transfection according to the manufacturer’s protocol.
Co-IP and GST pull-down Assay - Cell extracts were
incubated with antibodies and Protein A agarose
(Pierce) overnight at 4C. Normal mouse or rabbit
IgG (Santa Cruz Biotech) was used as negative
control. After washed for five times with lysed buffer
(RIPA, Beyotime, P0013), the resulting beads were
eluted with the 2x SDS sample buffer by boiling for 8
min at 100C, and the samples were analyzed by
Western blots. GST alone, GST-tagged and His-tagged
proteins were expressed in E.coli BL21. GST-tagged
proteins were purified by glutathione-Sepharose 4B
beads (Amersham Biosciences) according to the
manufacturer’s instructions (Amersham Biosciences).
His-tagged proteins were prepared and purified using
Ni-affinity resins (Merk). His-PIM2 protein was
mixed with GST or GST-PKM2 fusion protein in PBS
binding buffer (Takara’s PBS, pH 7.4) at 4C for 2hr,
followed by the addition of 20l of glutathione-
sepharose 4B beads. After 1hr of incubation with
nutation, the beads were washed with PBS for five
times. The resulting beads were eluted with the 2x
SDS sample buffer and analyzed by Western blots
Confocal Immunofluorescence Microscopy - Cells
were plated into 6-well plates with a density of 8×104
cells per well. About 36hr after transfection, cells
were fixed with 4% paraformaldehyde for 20 min,
washed for three times with PBS, and treated with
0.2% Triton X-100 for 20 min followed by another
three washes with PBS. The cells were blocked with
3% BSA/PBS for 1hr followed by incubation with
primary antibody (1:100 anti-HA; 1:100 anti-FLAG)
for 2 hr at room temperature. After extensive wash,
cells were incubated with a secondary antibody
conjugated with goat anti-mouse IgG/TRITC antibody
for 1hr and counterstained with DAPI for 10 min. The
resulting signals were visualized by a confocal laser-
scanning microscope (OLYMPUS BX61).
In vitro Kinase Assay - For in vitro kinase assays,
PKM2-WT or PKM2-T454A recombinant proteins
were incubated with PIM2 in kinase buffer (20mM
MOPS, pH 7.4, 150mM NaCl, 12.5mM MgCl2, 1mM
MnCl2, 1mM EGTA, 1mM DTT, 10 uM ATP) (Sigma).
The reaction mixtures were incubated at 37C for 30
min (29). Aliquots of reaction mixtures were analyzed
by Western blots using rabbit anti-Phospho-Threonine
Luciferase Reporter Assays - Cells were seeded onto
6-well plates, transfected with PKM2-WT or PKM2-
T454A together with the reporter plasmid p2.1(30)
and control pSV40-Renilla, and exposed to either 20%
or 1% O2 for 24hr. Cell lysates were analyzed using
the Dual-Luciferase Assay
according to the manufacturer’s instructions (31,32).
Glucose Consumption and Lactate Production - Cells
were seeded onto 6-well plates and transfected with
plasmids or siRNAs. About 48 hours after transfection,
cells were washed and cultured in serum-free DMEM
for about 16 hr (12). Glucose levels in medium were
measured using a glucose assay kit (Sigma), and
lactate levels in medium were measured using a
Lactate assay kit (CMA, Microdialysis). These
readouts were normalized to corresponding protein
Cellular Oxygen Consumption Rate - Oxygen
consumption rate was analyzed using a Seahorse
XF24 Extracellular Flux Analyzer by real-time
monitoring mitochondria respiration. Cells were
plated onto XF24 cell culture plates (Seahorse
Bioscience) at a density of 2x104
incubated for 24 hr in a normal incubator. Then, cells
were equilibrated with bicarbonate-free buffered
DMEM medium in a 37C incubator for 60 minutes
without CO2 immediately before XF assay. Substrates
or perturbation compounds were prepared in an
identical assay medium as in the corresponding well
and were injected from the reagent ports automatically
to the wells at the designated time-points. The first
compound injected was oligomycin, which inhibits
complex V in the electron transport chain and causes a
decrease in respiration. The second was FCCP, which
drives mitochondrial respiration to its maximal
capacity, referred as the total reserve capacity. Finally,
Antimycin/Rotenone was injected to inhibit oxidative
phosphorylation at complex I and to block all
mitochondrial oxygen consumption (33).
Cell Proliferation Analysis - Cells were seeded onto 6-
well plates, transfected with PKM2-WT or PKM2-
cells per well and
T454A. After 24hr 1x104 cells were harvested and
seeded in triplicates onto 24-well plates, and cell
numbers were counted every 24 hr over a four-day
14CO2 Release Assay - To determine 14CO2 release,
cells were incubated in glucose free medium
containing 1 Ci/mL of either 6-14C-glucose or 1-14C-
glucose for 30min at 37C. Phenylethylamine was
used to absorb the released CO2. The radioactivity of
counting of phenylethylamine for three times with
30min intervals. This method is designed according to
the principle and protocol reported previously (34).
The radioactivity was normalized to cell numbers.
Quantitative Real-Time PCR - Total RNA was isolated
using a TRIzol kit (Omega), and cDNA was
synthesized using a cDNA synthesis kit (Takara).
Quantitative real-time PCR was performed using the
SYBR Green PCR Master Mix (Takara) on the Roche
480 system (Roche). The primers used in this study
were listed in Table S2.
Statistical Analysis - We determined the significance
of differences using Pearson's correlation test and
Student's t test (two-tailed). P<0.05 was considered to
PIM2 is a novel binding partner of PKM2 - To
better understand the regulation of PKM2, we
screened cDNA libraries of human brain, liver and
kidney by yeast two-hybrid using a C-terminal portion
(aa354-531) of PKM2 as the bait, since the N-
terminus of PKM2 tends to form tetramers (35). Fifty-
eight positive clones were identified from the screens
(Table S1). Among them, five clones encoded the
serine/threonine kinase PIM2 were identified from the
human kidney cDNA library. As shown in Fig. 1a, the
interaction between PKM2 and PIM2 was then
validated by independent yeast two-hybrid. To further
analyze their interaction, we transiently over-
expressed HA-tagged PKM2 and Flag-tagged PIM2 in
HEK293T cells. By co-immunoprecipitation (Co-IP)
analysis, we showed that PKM2 could pull down
PIM2 (Fig. 1b) and vice versa (Fig. 1c), although the
latter is weak. These results demonstrate that PIM2
and PKM2 interact with each other.
PKM2 consists of four domains: N, A1, B, A2
and C (Fig. 1d). To determine which domain(s) of
PKM2 bind to PIM2, we generated a series of HA-
tagged truncation mutants of PKM2 (Fig. 1d), and
tested their binding affinity with Flag-tagged PIM2 in
HEK293T cells by Co-IP. As shown in Fig. 1E, full-
14CO2 was quantified by scintillation
length PIM2 could interact with all the fragments of
PKM2 with various affinities, suggesting the presence
of multiple interaction surfaces. However, its
interaction with the amino acid sequence 219-531 of
PKM2 was much stronger than any other fragments,
consistent with our yeast-two hybrid results (Fig. 1e).
Furthermore, to determine whether PIM2 can directly
interact with PKM2, we performed GST pull-down
assays using purified recombinant His-tagged PIM2
and GST-tagged PKM2. As shown in Fig. 1f, GST-
tagged PKM2 could efficiently and specifically pull-
down His-tagged PIM2, suggesting PIM2 can directly
interact with PKM2.
Since PIM2 is highly homologous to PIM1 and
PIM3, we examined whether PKM2 could also
interact with PIM1 and PIM3. Interestingly, over-
expressed PKM2 interacted with PIM3 even stronger
than with PIM2, but failed to interact with PIM1 (Fig.
2a). In addition, as PKM1 is an alternative splice
product of the PKM gene (36), we asked whether
PKM1 could also interact with PIM2. As shown in Fig.
2b, over-expressed PKM1 bound to PIM2 in
HEK293T cells as measured by Co-IP. Moreover, to
determine whether endogenous PIM2 interacts with
PKM2, we performed Co-IP experiments with cell
lysates from HeLa or A549 cells. Using an anti-PKM2
antibody, we show that endogenous PKM2 could Co-
IP endogenous PIM2 in A549 cells (Fig. 2c) or HeLa
cells (Fig. 2d). Conversely, endogenous PIM2 could
Co-IP endogenous PKM2 in A549 cells (Fig. 2e).
microscopy analyses showed that PIM2 partially
overlapped with PKM2 in the nuclei of HeLa cells
(Fig. 2f). Taken together, our results demonstrate that
PIM2 is a novel binding partner of PKM2.
PIM2 phosphorylates PKM2 on Thr-454 -
PIM2 has been shown to regulate the functions of
proteins, such as BAD (37), p21 (29), p27(24) and 4E-
BP1 (38) via direct phosphorylation. We then
examined whether PIM2 could also phosphorylate
PKM2. For that purpose, we co-transfected Flag-
tagged PIM2 (vector, wild type or kinase dead PIM2)
with HA-tagged PKM2 into HEK293T cells.
Compared to the kinase dead PIM2 or vector control,
transfection of wild type PIM2 caused an increase in
PKM2 phosphorylation on threonine residues, as
detected by immunoblotting with a phospho-Thr
specific antibody (Fig. 3a). PIM2 did not affect the
phosphorylation levels of PKM2 on serine residues
(Fig. 3b), while as shown in Fig. 3c, it increased the
serine phosphorylation of the known PIM2 kinase
substrate, BAD (37), under the same condition.
To determine whether PIM2 could directly
phosphorylate PKM2, we performed in silico analyses
for potential PIM substrate motifs in PKM2 (38). As
shown in Fig. 3d, we identified two putative PIM
phosphorylation sites (Thr-405 and Thr-454) in PKM2.
To determine which site(s) could be phosphorylated
by PIM2, we generated phosphorylation-defective
mutants by mutating the threonine residue to alanine
(T405A or T454A). Interestingly, in HEK293T cells
mutation of Thr-405 had no effect on threonine
phosphorylation of PKM2, while mutation of Thr-454
phosphorylation (Fig. 3e), suggesting that the Thr-454
residue of PKM2 was targeted by PIM2 for
phosphorylation. In vitro kinase assays further
confirmed that PIM2 could directly phosphorylate
PKM2 on the Thr-454 residue. As shown in Fig. 3f,
recombinant wild type PIM2 increased the threonine
phosphorylation of wild type PKM2 by about five
fold, while it had no effects on the PKM2-T454A
mutant. These results strongly demonstrate that PIM2
directly phosphorylates PKM2 on the Thr-454 residue.
PIM2 positively regulates PKM2 protein
levels - Posttranslational regulation of PKM2, such as
phosphorylation or acetylation, has been reported to
regulate PKM2 function via affecting its protein
stability, translocation and homodimerization (14).
Thus, we hypothesized that T454 phosphorylation of
PKM2 by PIM2 regulates PKM2 protein stability. To
this end, PKM2 protein levels were analyzed when
HEK293T cells were co-transfected with Flag-tagged
PIM2 and HA-tagged PKM2. Compared to the vector
control, over-expression of PIM2 caused an increase
in both over-expressed and endogenous PKM2
proteins in HEK293T cells (Fig. 4a). Similarly, over-
expression of PIM2 also increased endogenous PKM2
protein levels in A549 cells (Fig. 4b). Conversely,
when PIM2 was knocked down by specific siRNA in
A549 (Fig. 4c) or HepG2 (Fig. 4d) cells, PKM2
protein levels was significantly reduced. However, the
mRNA levels of PKM2 was not significantly changed
when PIM2 was over-expressed or knocked down
(Fig. 4e), suggesting PIM2 regulates PKM2 protein
stability. To determine whether PIM2 stabilizes PKM2
via Thr-454 phosphorylation, the kinase dead (KD)
form of PIM2, K61A (37), was used. Compared to the
vector control, transfection of wild type PIM2 led to
an increased levels of over-expressed PKM2, while
kinase dead PIM2 had little effect on or slightly
decreased PKM2 levels in HEK293T cells (Fig. 4f).
Together, these data suggest that PIM2-mediated
phosphorylation of PKM2 on the Thr-454 residue
controls the abundance of PKM2 proteins.
PIM2 promotes PKM2-dependent
glycolysis and reduces mitochondrial respiration - A
recent study has shown that PIM1 and PIM3 regulate
energy metabolism and cell growth (39). Since PIM2
interacts with PKM2, which regulates glycolysis in
cancer cells, we hypothesize that PIM2 regulation of
glycolysis depends on PKM2. Indeed, over-expression
of PIM2 increased glucose consumption in both
HEK293T (Fig. 5a) and HepG2 (Fig. 5b) cells.
Moreover, over-expression of PIM2 also increased
lactate production in those cells (Fig. 5c and 5d).
These data suggest PIM2 regulates glycolysis in these
cells. To determine the role of endogenous PIM2 in
glycolysis, we knocked down PIM2 using specific
siRNA. As shown in Figs. 5a-d, PIM2 knockdown
decreased both glucose consumption and lactate
production in HEK293T and HepG2 cells, further
confirming the role of PIM2 on glycolysis. Next, we
asked whether PKM2 was required for PIM2
regulation of glycolysis. For that purpose, we first
knocked down PKM2 in HEK293T or HepG2 cells
using siRNA specifically targeting PKM2, and then
over-expressed Flag-tagged PIM2. As shown in Figs.
5a-d, both glucose consumption
production were no longer increased when PKM2 was
knocked down, suggesting PKM2 is indeed required
for PIM2-induced glycolysis in cancer cells.
To determine whether PIM2 stimulation of
glycolysis depends on PKM2 phosphorylation at the
Thr-454 residue, we transfected HEK293T cells with
HA-tagged wild type or T454A mutant PKM2. As
shown in Fig. 5e, over-expression of the T454A
mutant PKM2 decreased both glucose consumption
and lactate production as compared to over-expression
of wild type PKM2. Interestingly, over-expression of
the T454A mutant PKM2 also resulted in a higher PK
enzyme activity (Fig. 5f). These data suggest that
PKM2 Thr-454 phosphorylation is involved in
regulating glycolysis and controlling its enzymatic
Changes in glycolysis may affect mitochondrial
functions. In order to determine whether PIM2
regulation of PKM2 influences
functions, we examined the metabolic flux by
Seahorse analysis and using 14C (1-14C and 6-14C) -
labeled glucose. As shown in Fig. 6a and Fig. 6b, the
mitochondrial respiration, as indicated by oxygen
consumption, was increased in cells expressing the
T454A-mutant PKM2. In contrast to wild type,
T454A-mutant PKM2 significantly increased the
release of 6-14CO2 from [6-14C] glucose (Fig. 6c),
while had no effects on the release of 1-14CO2 from [1-
14C] glucose (Fig. 6d). Thus, the ratio of 1-14CO2 to 6-
14CO2 was decreased (Fig. 6e). Together, our results
suggest that PKM2 phosphorylation at Thr-454 by
PIM2 reduces mitochondrial respiration.
promotes its cofactor functions - Previous studies have
reported that PKM2 can function as a transcriptional
cofactor to stimulate HIF-1 and -catenin-mediated
gene transcription (9,10). To determine whether
PIM2-dependent phosphorylation of PKM2 on Thr-
454 affects its transcriptional cofactor functions, we
first examined the effects of wild type and T454A
mutant PKM2 on hypoxia-induced gene transcription.
For that purpose, we co-transfected HEK293T cells
with HA-tagged PKM2 (wild type or T454A mutant)
with the p2.1 plasmid, a luciferase reporter construct
containing HREs of the HIF-1 target gene ENO1 (30)
and pSV40-renilla as the control for transfection.
After 24hr of culture, cells were incubated in either
20% or 1% O2 for another 24 hr. As shown in Fig. 7a,
wild type PKM2 could strongly activate the promoter
under the hypoxic condition, while T454A mutant
PKM2 was about as half active as wild type PKM2 on
HIF-1-mediated transcription. Moreover, T454A
mutant PKM2 was significantly less potent to activate
endogenous HIF-1 target genes, such as LDHA,
PDK1, ENO1, VEGF and GLUT1, in HEK293T,
HepG2 or HeLa cells (Fig. 7b-d). Similarly, T454A
mutant PKM2 was also significantly less potent to
activate endogenous -catenin target genes, such as
Myc and CCND1, in HEK293T, HepG2 or HeLa cells
(Fig. 7e-g). These results demonstrated that
phosphorylation of PKM2 on Thr-454 is important for
its co-activator functions on HIF-1 and -catenin.
increases cancer cell proliferation - To determine
whether phosphorylation of PKM2 on Thr-454
regulates proliferation of cancer cells, we transfected
A549 cells with HA-tagged PKM2 (wild type or
T454A mutant). As shown in Fig. 8a, cells transfected
with T545A mutant PKM2 grew significantly slower
than those transfected with wild type PKM2,
suggesting phosphorylation of PKM2 on Thr454 is
required to promote cancer cell proliferation. Previous
studies have shown that the ratio of 1-14CO2/6-14CO2
reflects the malignant degree of tumors (40). In our
study, the ratio of 1-14CO2/6-14CO2 was lower in the
presence of T454A mutant than wild type PKM2 (Fig.
6e), suggesting that Thr-454 phosphorylation of
PKM2 is required for malignant phenotype of cancer
Reprogramed energy metabolism is a hallmark of
cancer cells. The Warburg effect, which is
characterized by an increased glycolysis even in the
presence of oxygen, is currently the best known
metabolic abnormality in cancer cells (1). Numerous
studies in recent years have demonstrated the PKM2
isoform of pyruvate kinase as a key regulator of the
Warburg effect (41). Supporting this notion, during
tumorigenesis PKM1, L and R isoforms of pyruvate
kinase are gradually diminished and replaced by
PKM2 (35). Moreover, it has been shown that
knockdown of PKM2 in cancer cells could decrease
the rate of glycolysis in addition to inhibition of cell
proliferation (36). Importantly, introduction of PKM2,
but not PKM1, could stimulate glycolysis and
promote tumorigenesis (36). Despite such extensive
studies on the oncogenic role of PKM2, the
underlying mechanisms of PKM2 regulation are still
not fully understood.
In this study, using a C-terminal portion (aa354-
531) of PKM2 as the bait in yeast-two hybrid, we
identified multiple clones encoded PIM2 from the
human kidney cDNA library. The interaction between
PIM2 and PKM2 was confirmed through independent
yeast two-hybrid and Co-IP of over-expressed or
endogenous proteins. GST pull-down data show that
recombinant PIM2 and PKM2 bind to each other,
suggesting PIM2 directly interacts with PKM2.
Interestingly, by Co-IP we found that PIM2 could
interact with all fragments of PKM2, which we tested,
with various affinities, suggesting a complicated
interaction between PIM2 and PKM2. However, it is
also likely that some of these interactions were
indirect and were mediated by other cellular proteins.
Nevertheless, consistent with our yeast-two-hybrid
data, a fragment covering aa219-531 of PKM2
displayed the strongest binding with PIM2.
On one hand, PKM2 is the alternative splicing
form of PKM1, and they differ by only 23 amino
acids. Thus, we predicted that PKM1 could also
interact with PIM2. Indeed, by Co-IP we show over-
expressed PIM2 binds to PKM1. Thus, it would not be
surprising if PIM2 also regulates PKM1 functions.
However, since PKM1 is often down regulated and
replaced by PKM2 in cancer cells, the relevance of
PKM1 regulation by PIM2 is probably not significant
at least in cancer development. On the other hand,
PIM2 belongs to a family of serine/threonine protein
kinase. The other members of this family include
PIM1 and PIM3. Thus, we also examined the
affinities of PKM2 binding to all three PIMs.
Interestingly, our Co-IP data show that PKM2 could
interact with PIM2 and PIM3, but not PIM1. In
addition, it appears that PIM3 has the strongest
interaction with PKM2. However, since PIM1, 2 and 3
are highly similar in structure (21), it is possible that
all three PIMs may phosphorylate PKM2 and regulate
its functions. Consistent with the data in this study,
PIMs are known proto-oncogenes (21). It would be
interesting to test whether the oncogenic functions of
PIM1 and PIM3 in cancer cells also require PKM2 in
Since PIM2 is a serine/threonine protein kinase
and PKM2 is a known phosphorylated protein, we
tested the hypothesis that PKM2 is a novel substrate
of PIM2. Previous studies have identified several
substrates of PIM2, including BAD (37), p21 (29),
p27(24) and 4E-BP1 (38). From these studies, a
consensus substrate motif for PIM2 has been
concluded (21). Interestingly, through in silico
analysis, we have identified two PIM2 substrate
motifs containing the potential phosphorylation at
Thr-405 and Thr-454. However, mutation analyses
and in vitro kinase assays demonstrated that only the
Thr-454 residue of PKM2 could be targeted by PIM2.
Thus, PKM2 is a new substrate of PIM2.
Post-translational modifications of PKM2 and
protein-protein interaction have been reported to
regulate its enzymatic activity or protein stability (41).
For example, glucose-stimulated acetylation decreases
PKM2 enzymatic activity and promotes CMA-
mediated degradation (14). In this study, we found
that cells transfected with T454A mutant PKM2
displayed higher pyruvate kinase activity than those
with wild type PKM2
phosphorylation of PKM2 inhibits its enzymatic
activity. In addition, our data suggest that PIM2
positively regulates the abundance of PKM2, and such
regulation is most likely post-translational, because
PIM2 does not affect PKM2 mRNA expression. Thus,
PIM2 functionally regulates PKM2.
A previous study has reported a stimulatory role
of PIM2 on glycolysis in cancer cells (42). However
the underlying mechanism of PIM2 regulating glucose
metabolism was not clear. Through loss of function
and gain of function analyses we show that PIM2-
dependent stimulation of glycolysis requires PKM2,
further suggesting the significance of their interaction.
Consistent with these data, we show that T454A
mutant PKM2 is less potent to promote glycolysis
than wild type, suggesting
phosphorylation on Thr-454 residue is critical for
PIM2/PKM2-induced glycolysis in cancer cells. In
addition to increased glycolysis, our data also show
that PIM2-mediated phosphorylation of PKM2
inhibits mitochondrial oxidative phosphorylation,
suggesting Thr-454 phosphorylation of PKM2 is
essential for the switch of glucose metabolism.
Previous study indicates that high pyruvate kinase
activity suppressed glycolysis and cell proliferation
(35). In our study we found T454A mutant PKM2 also
had higher PK catalytic activity than wild type. In the
mean time, glycolysis and cell proliferation were
down-regulated. It probably T454A mutant promotes
tetramer formation of PKM2 and suppresses
tumorigenesis which is consistent with previous study
Besides its role in glycolysis, PKM2 has recently
been reported as a transcriptional co-activator for
oncogenic transcription factors, such as HIF-1 (9)
and -catenin (10). Thus, we tested whether Thr-454
phosphorylation of PKM2 affects the cofactor
functions of this protein. Indeed, our results
demonstrated that T454 mutant PKM2 is less potent to
activate both HIF-1 and -catenin target gene
expression than wild type, suggesting PIM2-mediated
phosphorylation of PKM2 affects broadly on PKM2
In summary, we have identified PIM2 as a novel
regulator of PKM2. Multiple approaches show that
PIM2 and PKM2 interact with each other.
Functionally, through phosphorylating the Thr-454
residue, PIM2 regulates several aspects of PKM2
functions in metabolic reprogramming of cancer cells
(Fig. 8b). Our results demonstrate that PIM2-
dependent phosphorylation of PKM2 is critically
involved in the Warburg effect in cancer cells. So far,
PIM2 has been extensively targeted for cancer therapy.
Several inhibitors of PIM2 are in clinical trials. Our
results will provide a novel mechanistic basis of
targeting PIM2 in treating cancer.
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*This study was supported by research grants from “973” Project (No.2012CB932604), New Drug Discovery
Project (No.2012ZX09506-001-005), Shanghai Leading Academic Discipline Project (No.S30203), National
Natural Science Foundation of China (No. 81071180, 81001008 & 81372195), Shanghai Pujiang Program
(No.13PJ1406000) and Science and Technology Commission of Shanghai Municipality (No.134119a5600).
1 School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, PR China.
2 Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
3 Department of Nuclear Medicine, Renji Hospital, Shanghai Jiao Tong University, School of Medicine,
Shanghai 200127, China.
4 Department of Medicine and Developmental & Molecular Biology, Albert Einstein College of Medicine, New
York, NY 10461, USA.
FIGURE 1. PIM2 is a novel binding partner of PKM2
a. Interaction between PKM2 (aa354-531) and PIM2 (aa182-311) was re-examined by yeast two-hydrid using
SD-Leu-Trp or SD-Leu-Trp-His-Ade culture medium. Vector pGADT7-T with pGBKT7-p53 or pGBKT7-Lam
was used as positive control and negative control, respectively.
b and c. Interaction between Flag-tagged PIM2 and HA-tagged PKM2 full-length proteins was examined by Co-
IP followed by Western blotting using anti-HA antibody or anti-Flag antibody. Tagged proteins were over-
expressed in HEK293T cells by transient transfection.
d. Schematic representation of PKM2 protein fragments used in the mapping analysis
e. Flag-tagged PIM2 was co-expressed in HEK293T cells with HA-tagged PKM2 fragments as indicated. Co-IP
followed Western blotting was performed to determine their interaction.
f. GST pull-down assays were performed to examine the direct interaction between PKM2 and PIM2 using
recombinant His-tagged PIM2 and GST-tagged PKM2.
FIGURE 2. PIM2 interacts with PKM2
a. HA-tagged PKM2 was co-expressed with Flag-tagged PIM1, PIM2 or PIM3 in HEK293T cells. Co-IP
followed by Western blotting was performed to determine their interaction.
B. Interaction between HA-tagged PKM1 and Flag-tagged PIM2 were examined by Co-IP followed by Western
c, d and e. The association of endogenous PIM2 and PKM2 in A549 (c and d) or HeLa (e) cells was analyzed by
Co-IP followed by Western blotting using anti-PIM2 antibody and anti-PKM2 antibody.
f. Confocal immunofluorescence microscopy was performed to analyze localization of PKM2 and PIM2 in
FIGURE 3. PIM2 phosphorylates PKM2 on Thr-454
a and b. HA-tagged PKM2 was co-transfected with empty vector, Flag-tagged PIM2-KD (kinase dead) or PIM2-
WT (wild type) in HEK293T cells. Two days after transfection, PKM2 proteins were immunoprecipitated using
anti-HA antibody, and HA-tagged PKM2 protein levels were normalized before phosphor-threonine (a) or
phosphor-serine (b) levels were detected by Western blotting using indicated antibodies.
c. Effects of PIM2 on phosphor-serine levels of HA-tagged BAD in HEK293T cells.
d. Identification of two putative PIM2 substrate motifs in PKM2. T405 and T454 residues were highlighted by
e. Effects of Flag-tagged PIM2 (KD or WT) on threonine phosphorylation of HA-tagged PKM2-T405A
(threonine 405 to alanine mutation) or T454A (threonine 454 to alanine mutation) in HEK293T cells.
f. In vitro kinase assay to determine the effects of recombinant PIM2 on threonine phosphorylation of His-
tagged PKM2-WT or T454A.
FIGURE 4. PIM2 regulates PKM2 protein levels
a. HEK293T cells were co-transfected Flag-tagged PIM2-WT (or empty vector as control) with HA-tagged
PKM2. Two days after transfection, the protein levels were analyzed by Western blotting using indicated
b. Effects of over-expressing PIM2 on PKM2 protein levels in A549 cells. A549 cells were transfected with
Flag-tagged PIM2. Two days after transfection, the protein levels were analyzed by Western blotting using
c and d. Effects of PIM2 knockdown by specific siRNA on PKM2 protein levels in A549 (C) and HepG2 (D)
e. Effects of over-expressing or knocking down PIM2 on PKM2 mRNA levels in 293T cells, HEK293T cells
were transfected with Flag-tagged PIM2 or siRNA-PIM2. Two days after transfection, the mRNA levels of
PKM2 were analyzed by qRT-PCR. (Data represent mean ± SEM (n = 3), *p<0.05.
f. Requirement of kinase activity of PIM2 in regulation PKM2 protein levels in HEK293T cells. HEK293T cells
were transfected with Flag-tagged PIM2 (WT or KD) or emporty vector as control. Two days after transfection,
the protein levels were analyzed by Western blotting using indicated antibody.
FIGURE 5. PIM2 promotes PKM2-dependent glycolysis
a and b. HEK293T (a) or HepG2 (b) cells were transfected with an empty vector or Flag-tagged PIM2-WT;
PKM2 or control -siRNA followed by Flag-tagged PIM2-WT; or PIM2 or control - siRNA. Two days after
transfection, the medium was replaced by serum free medium for another 12-16 hr cultured. Glucose levels in
medium were examined.
c and d. Same treatments as in a and b. Lactate levels in medium were examined.
e. A549 cells were transfected with either HA-tagged PKM2-WT or T454A. Two days after transfection, the
medium was replaced by serum free medium for another 12-16 hr cultured. Glucose and lactate levels in
medium were examined.
f. A549 cells were transfected with either HA-tagged PKM2-WT or T454A. Two days after transfection,
Pyruvate kinase activities in cell lysates were examined.
All data represent the means ± SEM of three independent experiments, *p < 0.05.
FIGURE 6. PIM2-phosphorylation of PKM2 regulates mitochondrial respiration
a-d. A549 cells were transfected with HA-tagged PKM2-WT or T454A. One day after transfection, cells were
re-plated into appropriate plates for analysis of O2 consumption (a) and OCR (b) by a Seahorse XF24
extracellular flux analyzer, 6-14CO2 (c) or 1-14CO2 (d) release from glucose.
e. Ratio of 1-14CO2 to 6-14CO2 from C and D.
All data represent the means ± SEM of three independent experiments, *p < 0.05.
FIGURE 7. Thr-454 phosphorylation of PKM2 promotes its cofactor functions
a. HEK293T cells were co-transfected with HA-tagged PKM2-WT or T454A, p2.1 and pSV40-Renilla.
Transfected cells were exposed to 20% O2 or 1% O2 for 24hr. The ratio of firefly to renilla luciferase activity
b - d. HEK293T (b), HepG2 (c) or HeLa (d) cells were transfected with HA-tagged PKM2-WT or T454A, Two
days after transfection, the cells were exposed to 1% O2 or 20% O2 for another 24hr. The mRNA levels of
indicated genes were examined by qRT-PCR.
e, f and g. HEK293T (e), HepG2 (f) or HeLa (g) cells were transfected with HA-tagged PKM2-WT or T454A,
Two days after transfection, the mRNA levels of indicated genes were examined by qRT-PCR.
All data represent the means ± SEM of three independent experiments, *p < 0.05.
FIGURE 8. Thr-454 Phosphorylation of PKM2 increases cancer cell proliferation.
a. A549 cells were transfected with HA-tagged PKM2-WT or T454A. One day after transfection, cells were re-
plated and analyzed for cell growth by counting cell number at the indicated time-points,
b. Summary of PIM2 regulation of PKM2 functions in cancer development.
All data represent the means ± SEM of three independent experiments, *p < 0.05.
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