Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway
Human Molecular Genetics
Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway
Li Lin 2 3
Chadwick M. Hales 1 2
Kathryn Garber 2 3
Peng Jin firstname.lastname@example.org 2 3
0 The Author 2014. Published by Oxford University Press. All rights reserved
1 Department of Neurology and Center for Neurodegenerative Diseases
2 Emory University School of Medicine , Atlanta, GA 30322 , USA
3 Department of Human Genetics
Polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. FTO is a nuclear protein and its physiological function remains largely unknown, but alterations in its expression in mice influence energy expenditure, food intake and, ultimately, body weight. To understand the molecular functions of FTO, we performed a yeast two-hybrid screen to identify the protein(s) that could directly interact with human FTO protein. Using multiple assays, we demonstrate that FTO interacts with three isoforms of calcium/calmodulin-dependent protein kinase II: a, b and g, which are protein kinases that phosphorylate a broad range of substrates. This interaction is functional; overexpression of FTO delays the dephosphorylation of cAMP response element-binding protein (CREB) in human neuroblastoma (SK-N-SH) cells, which in turn leads to a dramatic increase in the expression of the CREB targets neuropeptide receptor 1 (NPY1R) and brain-derived neurotrophic factor (BDNF), which already are known to regulate food intake and energy homeostasis. Thus, our results suggest that FTO could modulate obesity by regulating the activity of the CREB signaling pathway.
Data from the World Health Organization (WHO) indicate that
at least 2.8 million people die each year as a result of being
overweight or obese (http://www.who.int), due to the increased risk
of several serious health conditions, particularly diabetes,
hypertension, cardiovascular disease, breathing difficulties during
sleep and some cancers (1,2). Although changes in lifestyle are
a root cause of the increased rates of obesity throughout many
populations, genetic variation influences the risk of obesity,
and several genome-wide association studies have sought the
identity of the culprit genes.
One consistent result of genetic association studies of body
mass index (BMI) and obesity is the fat mass and
obesityassociated (FTO) gene (3 – 5). This well-conserved gene is
present in a single copy in vertebrates and is broadly expressed
with particular abundance in the brain and hypothalamus
(6 – 9). Data from mouse models support the role of FTO in the
regulation of body mass. Global loss of Fto in mice leads to a
significant reduction in weight and fat mass compared with
wildtype mice. Although Fto null mice eat more than their wild-type
counterparts, they also expend more energy (10). Mice that
overexpress Fto, on the other hand, have increased bodyweight and
fat mass resulting primarily from significantly increased food
intake in the absence of changes in energy expenditure and
physical activity compared with wild-type mice (11).
The physiological role of FTO is only beginning to be
revealed. Bioinformatics analysis indicated that FTO is a member
of the AlkB family of non-heme Fe (II)/2-OG-dependent
oxidative DNA/RNA demethylases (12). N6-methyladenosine (m6A)
is a major physiological substrate of FTO. FTO efficiently
demethylates abundant m6A residues in RNA but has low
activity to methylated thymidine (3-meT) and uracil (3-meT) (13).
However, how m6A modification is linked to obesity remains
to be determined.
In order to better understand the role of FTO in the regulation
of body mass, we sought binding partners for FTO using yeast
two-hybrid screens. We discovered an interaction between
FTO and Calcium/calmodulin-dependent protein kinase II
(CaMKII) by yeast hybridization that was confirmed by
glutathione S-transferases (GST) fusion protein pull-down assays
and by co-immunoprecipitation (co-IP). Furthermore, when
we overexpress FTO in SK-N-SH cells, the interaction of FTO
with CaMKII delays cAMP response element-binding protein
(CREB) dephosphorylation, which, in turn, enhances the
expression of neuropeptide receptor 1 (NPY1R) and brain-derived
neurotrophic factor (BDNF). The RNA demethylase activity of
FTO is at least partially required for this effect, as mutant FTO
without this activity partially abolishes this induced expression.
Overall, our study illustrates a new mechanism by which FTO
SKAR, CaMKIIg, RFX2 and Dlgap3 are candidate
interactors of FTO
To identify the interacting partners of Fto, a human brain cDNA
library was screened with a pGBT9-Fto bait construct. Out of 22
positive clones that were sequenced, four candidates were
identified: SKAR, RFX2, CaMKIIg and Dlgap3 (Fig. 1A). In
particular, CaMKII isoforms (both beta and gamma) were identified 16
times. To confirm their interactions with FTO, we made a
GST-FTO fusion protein to see if it bound to the candidates in
pull-down assays. The GST-FTO fusion protein was purified
using glutathione-sepharose beads, then incubated with in
vitro translated SKAR, CaMKIIg, RFX2 or Dlgap3. GST-tag
alone was included as a negative control. All four candidates
bound to FTO in this assay (Fig. 1B), confirming the interactions
suggested by the two-hybrid screen.
Interaction of CaMKIIg and FTO in vivo identified by co-IP
Because the prior experiments were performed in vitro, we next
sought to demonstrate that they occur in vivo, using co-IP
experiments. Plasmids encoding a FLAG-tagged FTO and an
hemagglutinin (HA)-tagged candidate interactor (SKAR,
CaMKIIg, RFX2 or Dlgap3) were co-transfected into human
embryonic kidney 293 (HEK293) cells. Anti-FLAG beads
were used for the IP, and the resulting proteins were eluted and
blotted with an anti-HA antibody. Of the four candidate
interactors, only CaMKIIg was immunoprecipitated in conjunction
with FTO. A reverse experiment in which an anti-HA antibody
was followed by a western blot with an anti-FLAG antibody
also indicated that CaMKIIg was the only candidate protein
that co-immunoprecipitated with FTO (Fig. 2A).
Multiple CaMKII isoforms interact with FTO
CaMKII has two additional isoforms, CaMKIIa and b. To
examine the breadth of interaction between CaMKII and FTO,
additional co-IP experiments were performed. The coding
region for each isoform was cloned into a pCDNA3 vector,
which adds a Myc tag to the inserted sequence. Plasmids
encoding FLAG-tagged FTO and a Myc-tagged CaMKII isoform were
co-transfected into HEK293 cells. FTO was immunoprecipitated
using anti-FLAG beads, and immunoblotting of the resulting
precipitate was performed with antibody to the Myc tag. The reverse
experiment was carried out by immunoprecipitating with the
anti-Myc antibody, followed by immunoblotting with the
antiFLAG antibody. These experiments consistently indicated that
all three forms of CaMKIIa, b and g co-IP with FTO (Fig. 2B).
CREB phosphorylation is regulated by FTO
To investigate the consequences of the interaction of FTO with
CaMKII, we first explored its role in phosphorylation status of
the two interacting proteins. CaMKII did not phosphorylate
fulllength recombinant mouse Fto (mFto) nor the C-terminal
domain (327 – 502 amino acids) alone (Fig. 3A). Neither did
excess full-length recombinant mFto affect CaMKII
phosphorylation activity significantly (Fig. 3B). Thus, we turned our
attention to another known substrate of CaMKII, CREB. We
assayed CREB phosphorylation in human neuroblastoma
(SK-N-SH) cells in which FTO was overexpressed. A lentivirus
containing Fto was prepared in HEK293 cells and used to infect
SK-N-SH cells. Forty-eight hours after infection, the cells were
serum-starved for 24 h, then stimulated with 20 mM of forskolin
for different time periods to stimulate neuronal differentiation.
Phosphorylation of CREB was measured by western blotting.
An increase in CREB phosphorylation was observed from the
initial time point of 5 min of forskolin stimulation (Fig. 4).
The effect of forskolin on CREB phosphorylation exhibited
different kinetics in the cells infected with lenti-Fto compared with
the lenti-GFP control. Whereas in the GFP expressing cells,
CREB phosphorylation was weakened with 30 min of forskolin
stimulation, it did not begin to go down until 60 min of forskolin
stimulation in Lenti-Fto infected cells (Fig. 4). These results
suggest that overexpression of FTO delays the
dephosphorylation of CREB.
Furthermore, to determine whether FTO-mediated
modulation on CREB phosphorylation depends on the FTO
demethylation activity, we performed parallel analyses using a previously
described mutant FTO protein in which two iron (II) ligands were
mutated (H231A, D233A), resulting in complete loss of
m6Ademethylation activity (13). We found that the mutant FTO
lacking m6A-demethylation activity did not delay CREB
dephosphorylation to the same extent as wild-type FTO, suggesting
that the m6A-demethylation activity of FTO is involved in the
FTO-mediated modulation of CREB phosphorylation.
Expression of BDNF and NPY receptor 1 are increased
in FTO-overexpressing cells
CREB phosphorylation can either stimulate or inhibit its target
genes by binding to the regulatory CREs (14). Because we
observed that the dephosphorylation of CREB was delayed in
the presence of excess FTO, we next asked whether the
expression levels of known CREB target genes were affected. Using
real-time quantification PCR, we examined three known
CREB target genes: NPY, NPY1R and BDNF. Whereas the
expression levels of NPY1R and BDNF were significantly
increased after 30 min of forskolin stimulation in Lenti-Fto
infected cells, expression levels of CREB and NPY were
unchanged (Fig. 5). Differences in the activation of NPY1R and
BDNF expression between Lenti-GFP and Lenti-Fto infected
cells are likely a result of the delayed dephosphorylation of
CREB that was demonstrated in the previous experiments.
Consistent with this notion, the expression of mutant Fto induced
significantly less expression of NPY1R and BDNF after 30 min of
forskolin stimulation (Fig. 5).
The association of FTO with obesity is well established. The
initial report identified a single-nucleotide polymorphism
(SNP) in the first intron of FTO (rs9939609) that was strongly
associated with type 2 diabetes and increased BMI in a UK
population (7). Following this report, associations between additional
SNPs in FTO and human BMI have been reported in various
populations (3,15 – 17). The link between FTO risk alleles and
obesity has been supported by both loss-of-function and
overexpression mouse models (10,11). The identification of m6A
residues in RNA as a physiological target that is oxidatively
demethylated has increased our understanding of the activity
of this protein (13), but the mechanism by which this influences
obesity has largely been unknown. We report here a functional
pathway that links FTO with obesity: FTO¼ .CaMKII¼ .
CREB¼ .NPY. Increased FTO expression delays CREB
dephosphorylation by interacting with CaMKII, resulting in
the increased gene expression of NPY1R and BDNF. These
two proteins, in turn, are already known to regulate food intake
and energy homeostasis, thereby connecting the pathway with
BMI and obesity.
CaMKII is a serine/threonine protein kinase family encoded
by four genes (a, b, g and d) in mammals (18). CaMKIIa and
b are highly expressed in neurons (19 – 22). CaMKII is regulated
by multiple phosphorylation sites that alter its enzymatic activity
and induce its interaction with other proteins (23). CaMKII
phosphorylates a broad range of substrates, which are involved in
numerous physiological pathways, including cell development,
proliferation, cellular transport and neuronal function (24 – 28).
CaMKII has also been reported to regulate hepatic glucose
homeostasis in obesity (29).
Interactions between signaling molecules and various
protein-binding partners are critical for cells to respond
appropriately to their physiological status. CaMKII is an essential
cellular kinase that binds to and phosphorylates a variety of targets
in response to stimuli. Numerous binding proteins of CaMKII
have been identified via binding or IP assays. For example,
CaMKII binds to and phosphorylates tau and amyloid precursor
protein, which are key proteins in the pathogenesis of
Alzheimer’s disease (AD) (30,31). At the same time, CaMKII
auto-phosphorylation is altered in the brains of AD patients
(32). CaMKII also binds to the cyclin-dependent kinase 5
activators p35 and p39 (33), suggesting that CaMKII is involved in a
signaling pathway that contributes to synaptic plasticity,
memory and learning. Targets of CaMKII may achieve a
functional effect in one of two ways; the interaction might alter the
kinase activity of CaMKII through an allosteric mechanism, or
it might direct CaMKII to its substrates, thereby leading to
faster and more efficient phosphorylation of key proteins. In
our case, we found that FTO did not alter the kinase activity of
CaMKII. Therefore, a plausible explanation is that binding of
FTO to CaMKII induces CaMKII to phosphorylate CREB, in
that way resulting in the prolongation of CREB phosphorylation.
CREB is a transcriptional activator and promotes
adipogenesis by regulating glucose homeostasis (34). It has been reported
that CREB can be phosphorylated by CaMKII (35,36).
PhosphoCREB, by binding to CRE, leads to stimulation or inhibition of
target genes, such as NPY and BDNF, indicating that CREB is
a regulator of NPY (37). NPY is a 36-amino acid peptide with
widespread distribution in the brain (38 – 41). It mediates food
intake and decreases physical activity by acting through its
receptors (40,42 – 44). Five known NPY receptors (Y1 through
Y5) have been found in mammals (45 – 47). It has been shown
that hypothalamic NPY expression is up-regulated by fasting,
and the blockade of NPY receptors leads to the inhibition of
NPYergic activity, and a resulting reduction in food intake
(48 – 51). BDNF also is regulated by CREB. BDNF is a
member of the neurotrophin family and is widely expressed
throughout the central nervous system and skeletal muscles
(52 – 55). BDNF plays a critical role in normal neural
development (56 – 61); knockout of BDNF in mice results in
developmental defects in the brain, and the mice die soon after birth
(62). BDNF regulates energy homeostasis through binding to
tropomyosin-related kinase B receptor (63 – 67). Central
injection of BDNF gradually reduces food intake and causes body
weight loss in rats (68,69). Genome-wide associated studies
found that SNPs in BDNF are associated with BMI and obesity
In our study, mRNA levels of NPY1R and BDNF were
significantly increased in FTO-overexpressing cells. The time at which
NPY1R and BDNF mRNA levels are maximal matches perfectly
with the prolongation of CREB phosphorylation that occurs in
conjunction with FTO overexpression. Thus, we suggest that
expression levels of NPY1R and BDNF are affected by CREB
through NPY and that FTO plays a role in obesity by regulating
the CREB signaling system. The RNA demethylase activity of
FTO contributes to this activity but does not fully explain the
effects we see; a mutant form of FTO has an intermediate
effect on CREB phosphorylation and induction of NPY1R and
BDNF expression compared with overexpression of wild-type
FTO and to the control samples.
In sum, we propose a cellular mechanism by which FTO
functions in obesity. The interaction of FTO with CaMKII triggers
the prolongation of CREB phosphorylation, which ultimately
affects the expression levels of at least two key genes that
regulate energy balance, BDNF and NPY1R. These results
provide new targets for the development of therapeutic
approaches to obesity.
MATERIALS AND METHODS
Full-length mFto complementary DNA was cloned using mouse
brain cDNA library. The coding region was then cloned into the
pGEX-2TK vector or the pCDNA3 vector or the Lenti-GFP
vector (with Flag tag). The full-length Fto coding region with
mutation at H231A and D233A (kindly provided by Dr Chuan
He at University of Chicago) was cloned into the Lenti-GFP
vector. Full-length cDNAs of SKAR, CaMKIIg, RFX2 and
Dlgap3 were cloned into the pCDNA3 vector (with HA tag).
Full-length CaMKIIa, b and g cDNAs were cloned into the
pCDNA3 vector (with Myc tag). The sequences of all constructs
were confirmed by DNA sequencing.
Yeast two-hybrid screen
The full-length mFto cDNA was fused in frame with the GAL4
DNA-binding domain of the pGBT9 vector to generate
pGBT9-Fto bait construct. Yeast two-hybrid screening service
was provided by Duke University Genomic Core. Briefly,
PGBT9-Fto construct was co-transfected with human cDNA
keratinocyte libraries in Saccharomyces cerevisiae. Positive clones
were identified by TRP1 selection at 0 mM 3-aminotriazol. All
positive clones were sequenced by Sanger sequencing. The
details of the pGBT9-Fto bait construction and yeast two-hybrid
screening protocols are available upon request.
GST fusion protein purification and GST pull down assay
BL21 (DE3) pLysS Competent Cells (Promega) were
transformed with GST constructs (both GST and GST-Fto), and
single colonies were grown in 10 ml of Lysogenic Broth (LB)/
ampicillin (100 mg/ml) overnight at 378C. 500 ml of LB were
inoculated with 10 ml of overnight culture at 378C with
shaking for 2 h until absorbance at 600 nm reached between
0.6. Culture was induced with 16 mM isopropyl
b-D-1-thiogalactopyranoside and grown at 288C for 6 h. Cells were pelleted by
centrifugation at 3000g for 15 min and washed twice with
ice-cold 1 × phosphate buffered saline (PBS). Cells were
resuspended in 1 ml of ice-cold Tris-buffered saline and 1 ml of
Profound Lysis buffer (Pierce) with 1 × Complete ethylene
diamine-tetra-acetic acid (EDTA)-free protease inhibitor complex
(Roche) and inverted to mix immediately. The cellular
resuspension was incubated on ice for 30 min. Lysate was
centrifuged at 12 000g for 10 min. Supernatant was collected and stored
at 2808C until use. Glutathione-sepharose beads
(AmershamPharmacia) were washed and resuspended in 1 × PBS, to make
a 50%-bead slurry. 200 ml of bead slurry was incubated with
200 ml bacterial lysate for 1 h at 48C. Beads were washed twice
with 1 × PBS with 0.5% Triton X-100. In a 1.5-ml tube, 200 ml
of GST/or GST-FTO beads were incubated with 22 ml of the
appropriate in vitro translated protein (SKAR, CaMKIIg, RFX2 or
Dlgap3; TnT Coupled Reticulocyte Lysate Systems, Promega)
diluted with 180 ml of ice-cold binding buffer (1 × PBS; 0.2%
Triton X-100; Protease Inhibitor) at 48C for 2 h. Beads were
then washed twice with 1 ml of binding buffer. The beads were
resuspended with protein loading buffer and denatured at 1008C
for 5 min. Supernatants were separated by 6% polyacrylamide
gel electrophoresis (PAGE) for analysis.
HEK293 cells were cultured and transfected with 10 mg of the
each relevant plasmid using Lipofectamine 2000 (Invitrogen)
on 10-cm plate, according to the instructions of the manufacturer.
After 48 h, cells were harvested and lysed in lysis buffer [50 mM
Tris – HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 10% glycerol and 0.5% Triton X-100 supplemented with
complete protease inhibitor cocktail]. Lysates were kept on ice for
30 min and cell debris was removed by centrifugation. Following
the addition of 50 ml Protein A Agarose (Invitrogen), supernatants
were incubated with mouse monoclonal anti-myc or anti-HA
antibody overnight at 48C. [Alternatively, supernatants were
incubated with anti-FLAG beads (Sigma).] The beads were pelleted
by centrifugation; the supernatant was removed, and the pelleted
beads were washed three times with 1 ml of washing buffer
[50 mM Tris – HCl (pH 7.5), 150 mM NaCl, 5% glycerol and
0.1% Triton X-100]. Proteins were eluted with 50 ml of protein
loading buffer for 5 min at 1008C. Inputs and co-IPs were
subjected to sodium dodecyl sulfate– PAGE and transferred to
polyvinylidene difluoride membranes (Millipore).
For analysis of the IPs, western blots carrying inputs and
immunoprecipitated samples were first incubated with primary
antibody (mouse anti-HA or mouse anti-FTO or mouse anti-MYC)
followed by appropriate horseradish peroxidase-conjugated
antibodies. Membranes were processed following the HyGLO
Quick Spray western blotting protocol (Denville). For loading
controls, membranes were stripped and re-probed with the
antibody against GAPDH (Ambion).
CaMKII activity assay
CaMKII activity assay was performed using CaM Kinase II
Assay kit (Upstate) following the manufacturer’s instructions.
To determine whether CaMKII can phosphorylate FTO, the
substrates were used 25 mM of purified recombinant full-length
mouse FTO or the C-terminal domain of mouse FTO. Bovine
serum albumin (BSA) was used as a negative substrate. The
positive control was a control peptide from the kit. HEK293 cells
were transfected with Myc-CaMKIIa, b and g and cells lysate
were immonuprecipitated with an anti-MYC antibody. The
Protein A Agarose and corresponding IPs were added to the
reactions. To determine whether FTO can affect CaMKII
phosphorylation activity, the IPed Protein A Agarose and 100 ng of
recombinant full-length mouse FTO or BSA were added to the
reactions. Peptide from the kit was used as the substrate.
Virus infection and forskolin treatment
Lenti-GFP, Lenti-Fto and Lenti-mutant-Fto viruses were
packaged according to the method described previously (72).
SK-N-SH cells were cultured and infected 72 h by Lenti-GFP or
Lenti-Fto viruses. After starving for 24 h in FBS free medium,
cells were treated with 20 mM of forskolin for 0, 5, 15, 30, 60
and 180 min, then media were removed and cells were harvested
in ice-cold PBS and separated into two parts, one for total RNA
purification and another one for western blotting.
Reverse transcription and real-time PCR
Total RNAs were purified using the standard TRIzol method
(Invitrogen). Reverse transcription was been carried out using
SuperScript III Reverse Transcriptase (Invitrogen) according
to the instructions of the manufacturer. Messenger RNA
real-time PCR assays were performed according to protocols
provided by the vendor as described previously (73). Relative
quantities were determined via the DDCt method (74). All
relative quantity calculations were calibrated to control samples.
Three biological replicates were assayed.
We would like to thank Dr Chuan He (University of Chicago) for
providing the Fto constructs.
Conflict of Interest statement. None declared.
This work was supported in part NIH grant NS051630 (P.J.).
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