Concurrent interactome and metabolome analysis reveals role of AKT1 in central carbon metabolism
Gupta et al. BMC Res Notes
Concurrent interactome and metabolome analysis reveals role of AKT1 in central carbon metabolism
Nutan Gupta 0
Shweta Duggal 0
Ajay Kumar 0
Najmuddin Mohd Saquib 0
Kanury V. S. Rao 0 1
0 Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg , New Delhi 110067 , India
1 Translational Health Science & Technology Institute , NCR, Biotech Cluster, Faridabad 121001 , India
Objective: Signal transduction not only initiates entry into the cell cycle, but also reprograms the cell's metabolism. To control abnormalities in cell proliferation, both the aspects should be taken care of, thus pleiotropic signaling molecules are considered as crucial modulators. Considering this, we investigated the role of AKT1 in central carbon metabolism. The role of AKT1 has already been established in the process of cell cycle, but its contribution to the central carbon metabolism is sparsely studied. Results: To address this, we combined the metabolomics and proteomics approaches. In accordance to our hypothesis, we found that the AKT1 kinase activity is regulating the levels of acetyl CoA through pyruvate dehydrogenase complex. Further, the decreased levels of acetyl CoA and dependency of acetyl CoA acetyl transferase protein on AKT1 kinase activity was also found to perturb the synthesis rate of palmitic acid which is a representative of fatty acid. This was analyzed in the present study using lipid labeling method through mass spectrometry.
AKT1; Interactome; Metabolic flux; Central carbon metabolism; Affinity purification
Cell proliferation is required for the normal functioning
of various tissues and perturbations in this phenomenon
culminates into diseases like cancer. To proliferate, the
cell requires signalling for its division as well as for
generation of cell mass for the division of a mother cell into
daughter cells. Both of these can be regulated at various
levels by the same signaling molecule [
]. So in our quest
to understand pleiotropic functions of AKT1 during
proliferation, we aim to identify whether it plays any role in
CCM. Thus in addition to helping the cells to progress
through G1 phase of cell cycle [
], does it also help the
cells to generate mass for division?
The serine-threonine kinase AKT is a key signalling
molecule regulating wide range of cellular functions [
]. Mammalian cells express AKT as a three homologous
isoforms (AKT1-3), having high degree of amino acid
sequence similarity. Apoptotic rates in AKT1 deficient
animal models increased thereby suggesting its role in
] while AKT2 exercises its control over
metabolism, especially in insulin responsive tissues .
AKT3, along with AKT1 is implicated in mediating cell
growth processes [
A stable HEK293 cell line over expressing AKT1 was
generated using GATEWAY technology, as described earlier
]. SILAC labelling was used to detect varied protein
amounts among samples under different conditions. It
relies on the metabolic incorporation of the given light
and heavy forms of amino acids in the protein. Drug
treatment experiment was performed as described earlier
Immunoprecipitation of AKT1 and Interacting Proteins
A dual-tag affinity purification system, employing
C-terminal Strep and HA tags, was used to facilitate cloning,
detection, and purification of AKT1 along with its
binding partners. Affinity purification using the HA tag was
performed as published earlier [
]. Another aliquot of
supernatant of lysed cells was used for affinity
purification utilising STREP tag (Additional file 1: Method S1).
Protein Digestion and Desalting for Mass
Spectrometry, Cation Exchange Protocol (P/N 4326752, AB Sciex),
protein identification by Mass spectrometry and data
processing in protein pilot were performed as described
Validating AKT1 interacters by western blotting
To validate AKT1 binding partners, a few proteins from
AKT1 interacters list were randomly selected for reverse
immunoprecipitation. The bait proteins were over
expressed (as performed for AKT1),
immunoprecipitated and analyzed by western blotting, using
antibodies against AKT1 and relevant baits (Additional file 1:
13C6‑glucose labelling and metabolite extraction
Methods employed for labelling HEK-293 cells (MK-2206
treated and untreated) with 13C6-glucose and subsequent
metabolite extraction is described in Additional file 1:
Metabolite Identification by Mass Spectrometry
The detailed procedure for metabolite identification
through mass spectroscopy is described in Additional
file 1: Method S1. The gradient profile for LC–MS/MS is
provided in Additional file 3: Table S1.
Lipid labelling and extraction
Duplicate sets of HEK-293 cells (approximately 70%
confluent) were treated overnight in the presence and
absence of MK-2206. Next day a set of untreated and
treated cells were trypsinized and collected as 0 h sample.
For labelling lipids, another set of cells were incubated
in 13C6-glucose labelled medium for 7 h, after which the
cells were collected and processed for lipid extraction.
Analysis for palmitic acid was performed using mass
spectrometry as described in Additional file 1: Method
Metabolite data analysis was performed as described in Additional file 1: Method S1
To understand whether AKT1 is playing any role in CCM,
we first used the proteomics approach. This involved over
expression of our target and its subsequent pull down so
as to identify its interacting partners. In another set of
samples, kinase activity of target was inhibited and was
similarly processed. Then the interacting partners in both
the conditions were compared to identify its kinase
activity dependent interactions.
Identification of AKT1 binding partners
Workflow to identify binding partners of AKT1,
overexpressed in HEK-293 cells, is as illustrated in Additional
file 2: Figure S1. To understand kinase dependent
interacters, an allosteric AKT inhibitor- MK-2206 was used
]. As described in the “Methods” section, Protein
lists for each treated and untreated samples for two
biological samples were generated. Filters employed to
generate the final list of AKT1 interacters are illustrated in
Additional file 2: Figure S2. Here onwards, only those
proteins which were identified in both the replicate
sets were considered for further analysis. Heavy to light
ratios representing differential metabolic incorporation
of SILAC labels in treated and untreated samples were
averaged at each merge step. A union file from all
replicate data sets was generated after incorporating the
mentioned filters, and the differential association values were
normalized with that of AKT1.
In an effort to remove nonspecific interactors coming
along with tag or affinity matrix, AKT1 was swapped with
GFP in same biological system, i.e. HEK293 cells. GFP
was considered since it is a non-human protein which
shows minimal nonspecific binding to human proteins
]. A total of 210 proteins were co-immunoprecipitated
as eGFP interacters, in the replicate experiments
(Additional file 3: Table S1). Of these, 139 proteins overlapped
with AKT1 interactome list (Additional file 4: Table S2)
and thus were removed as possible non-specific
interacters. Finally, 904 unique interacters of AKT1
(Additional file 5: Table S3) were obtained at 95% confidence,
and 1% accepted G-FDR-fit (Additional file 2: Figure S7).
As an added measure to cross examine the obtained
interacters to be true AKT1 binding partners, few
random interacting proteins-BUB3 and GRB2, were reverse
immunoprecipitated and analyzed by western blot and
these were found to be true interacting partners of AKT1
Pathway enrichment of AKT1 interacters
The WebGestalt tool was used for KEGG analysis to
understand the functions of AKT1 interactors [
Swissprot-accession IDs of the identified interactors were
submitted for KEGG analysis at a stringent significance level
of ≤ 10−5. Metabolism mainly comprising of CCM
proteins was the most represented functional class. Other
significantly represented functional classes included
protein synthesis machinery, amino acid metabolism,
signaling pathways, pathways in cancer, regulation of actin
cytoskeleton, endocytosis/phagocytosis, cell cycle, etc.
Details are described in Additional file 2: Figure S3.
Effect of AKT1 inactivation on its binding partners
Treatment with MK-2206 results in complete loss of
signal for S473-specific AKT1 antibody and does not affect
total AKT1 protein concentration (Additional file 2:
Figure S4). This ensures that perturbations in
association of interacting partners are due to the diminishing
kinase activity and not due to the difference in protein
Out of 904 AKT1 interactors, 139 proteins (15.4%)
showed perturbed association with AKT1-(Additional
file 5: Table S3), i.e. these associations are selective for the
kinase active form of AKT1. Two-fold change was
considered to be significant change.
Kinetic flux profiling of the cell and metabolic characterization
Metabolomics approach was utilised to understand how
AKT kinase activity is regulating the levels of metabolites
belonging to CCM. The kinetic flux profiling was done
for quantitation of cellular metabolic flux in the cultured
cells. In these experiments, AKT1 over expressing cells
(Untreated control cells and the MK2206 treated cells)
were fed with uniformly labeled 13C6 glucose media.
The 14 metabolites representing the glycolytic pathway,
pentose phosphate pathway and the citrate cycle were
monitored for the incorporation of 13C6 isotopomer and
consumption of 12C isotopomer (Table 1). Specimen
chromatograms are represented in Additional file 2:
Figure S8. Additional file 6: Table S4 represents the relative
incorporation of 13C isotopomer and relative
consumption of 12C isotopomer in all four biological replicates.
As the percentage of the 13C labelled metabolite pool
increased, a concomitant reduction in the 12C
isotopomeric population ensued. These two-parallel
phenomenons kept the net concentration of metabolite pools
constant as described in Additional file 2: Figure S5.
Additional file 2: Figure S6 depicts possible labelling
pattern of metabolites when labeled 13C6 glucose media is
added to the culture.
G6P glucose-6-phosphate, FBP fructose-1,6 bis-phosphate, DHAP dihydroxy acetone phosphate, 3PG/2PG 3/2-phosphoglycerate, PEP phosphoenolpyruvate, AcoA
acetyl-CoA, OXA oxaloacetate, R5P ribulose-5-phosphate, IMP inosine monophosphate, AKG alpha-ketoglutarate
Synthesis rate of each metabolite was calculated
(experimental procedures) and correlated with
association values of proteins (with AKT1) mediating the
respective reactions. The levels of acetyl Co-A was found
to decrease when AKT kinase activity was diminished.
Acetyl CoA—a molecule connecting glycolysis and TCA
has two pools in cell—the mitochondrial pool generated
by PDHB using pyruvate as a substrate and cytoplasmic
pool generated by citrate lyase using citrate as a
substrate. In present study- metabolomics approach is not
sufficient to distinguish between these two pools. Thus
association of two enzymes generating them was
investigated with AKT1. Association of citrate lyase was not
found to be dependent on AKT1 kinase activity while the
association of PDHB was found to show decreased
association with AKT1. Hence these preliminary experiments
suggest that AKT1 kinase activity is regulating
acetylCoA levels through PDHB. Further we investigated the
effect of decreased concentration of acetyl CoA.
Association of ACAT2, (which converts acetyl-CoA to
acetoacetyl-CoA) with AKT1 was found to be decreased, when
AKT1 kinase activity was diminished. This may be due to
availability of less substrate. Proteins showing perturbed
association with AKT1 inhibition and their
corresponding reaction steps are highlighted in Additional file 2:
The experiment was conducted in duplicates for each condition and four separate peaks were tracked depicting 12C and 13C incorporation respectively
Effect of AKT kinase activity inhibition on lipid synthesis
Decreased synthesis rate of acetyl-CoA and depleted
association of PDHB and ACAT2 with AKT1 in MK-2206
treated cells suggests role of AKT1 in regulating lipid
synthesis. To validate this, rate of palmitic acid
synthesis (as a representative fatty acid) was determined, and
was found to decrease in presence of MK2206 (Table 2).
Chromatograms for this are represented in Additional
file 2: Figure S9.
Cell proliferation manifests in the increase in cell
number by an increase in the growth (i.e. increase in cell
mass) and the cell division. To accomplish this cells are
guided by molecular sentinels called “receptors” which
receives signals from the outside world and helps the cell
to decide accordingly. Cells have a smart arrangement of
molecular players viz protein kinases and phosphatases,
which implement the response to these signals. In our
study we focused on central carbon metabolism, as it
provides energy in the form of ATP and NADPH, which
is crucial for every cellular activity to happen. Similar
findings were also reported by other groups where
metabolic phenotype in AKT2 deficient mice with prominent
beta-cell dysfunction changed to type 2 diabetes because
of the presence of only a single functional allele of AKT1
]. Such findings suggest the complexity of regulatory
mechanism of AKT isoforms. In a systematic analysis of
breast cancer genes for physical interactions with their
interacting protein partners, PDHB was identified as a
direct interacter of AKT1 [
] and similar proteins were
found to interact in our study also. Our study though is
not sufficient to understand whether the interaction is
direct or indirect, since pulldown technique does not
discriminate between these two interaction modes.
MK2206 is a specific allosteric inhibitor of pan AKT
which do not perturb activity of any other kinase in a cell
]. Since isoform specific inhibitors are not
available, we over expressed and pull down only our target
to understand kinase specific perturbation in interacting
partners of AKT1 and not the other isoforms. But using
this approach, inhibition of basal levels of AKT2/3 may
have a impact on the outcome of the results. Moreover
over expression of AKT1 is changing its local
concentration which may result in more interacters since
interactions in cells are defined by their local concentration. But
it clearly indicates that these interactions are possible in
some physiological conditions.
Additional file 1: Methods S1. This document provides detailed
information on the materials used and protocols followed during the
Additional file 2: Figure S1. Detailed workflow to identify interacting
partners of AKT1 followed by their subsequent analysis. Figure S2. Filters
employed to generate the final list of AKT1 Interactors. Figure S3. KEGG
pathway enrichment for AKT1 binding partners using WebGestalt. Figure
S4. Validating the effect of AKT1 inhibitor (MK-2206) on AKT1 activity by
western blotting. Figure S5. Specimen depiction of relative incorporation
of labeled carbon in G6P and FBP metabolites in MK-2206 treated and
untreated samples. Figure S6. Possible labeling pattern for key metabolite
upon feeding cells with 13C6 Glucose. Figure S7. Specimen
chromatograms depicting estimated false discovery rates in Lys C and Trypsin
digested MK-2206 treated (Lys8Arg10) and untreated (Lys0Arg0) samples.
Figure S8. Specimen chromatograms showing metabolite peaks in the
untreated and MK-2206 treated samples for five different metabolites at
one of the targeted time points. Figure S9. Chromatograms are
corresponding to ion spectra of palmitic acid (as standard) and free fatty acids.
Figure S10. Schematic diagram of the CCM pathways depicting uptake
of glucose by the cells and its subsequent utilization across different
metabolites. Proteins showing perturbed association with AKT1 inhibition
and their corresponding reaction steps are highlighted in red colour in
Additional file 3: Table S1. Gradient profile for LC–MSMS method on
Agilent Polaris 5NH2 2 * 150 mm.
Additional file 4: Table S2. Sheet1: Consolidated union file generated
by integrating replicate data sets from the eGFPpull-down samples after
LC–MS/MS analysis. Sheet2: Represent the common interacting partners
between AKT1 and eGFP.
Additional file 5: Table S3. Sheet 1: Consolidated list of proteins
identified as AKT1 interactors from AKT1 pull down samples after targeting HA
and Strep tags. Interacting partners of AKT1 were analyzed by LC-MS/
MS in duplicate and proteins identified in both replicate sets were
only included to generate a union file at 1% global FDR (from fit); 95%
confidence. Sheet 2: Represents the list of interacting partners showing
perturbed association with AKT1 after inhibiting its kinase activity. Any
protein with a heavy: light ratio of SILAC labels greater than 2and less than
0.5 was considered perturbed.
Additional file 6: Table S4. Consumption of 12C isotopomeric form in the
targeted metabolites and incorporation of 13C in the targeted metabolites.
SILAC: stable isotope labeling with amino acids in cell culture; HA:
hemagglutinin; CCM: central carbon metabolism; GFP: green fluorescent protein;
FDR: false discovery rate; S473: serine 473; eGFP: enhanced green fluorescent
protein; TCA: tricarboxylic acid; PDHB: pyruvate dehydrogenase; ACAT2: acetyl
CoA acetyl transferase; ATP: adenosine triphosphate; NADPH: nicotinamide
adenine dinucleotide phosphate.
Conceived and designed the experiments: NG and KVSR. Performed the
experiments: NG, SD, AK. Mass spectrometry: SD and NMS. Contributed
reagents/materials/analysis tools: AK, SD and NMS. Analysed the data: NG, KVSR.
Contributed to the writing of the Manuscript: NG, SD, AK and KVSR. All authors
read and approved the final manuscript.
Authors thank Khushboo Adlakha for her assistance in acquiring metabolite
The authors declare that they have no competing interests.
Availability of data and materials
The data set supporting the conclusion of this article is available in the
“Proteome Xchange repository via the PRIDE database. It is publically available
via ProteomeXchange with identifier ProteomeXchange: PXD005976 (https
://www.ebi.ac.uk/pride/archive/projects/PXD005976). All relevant data are
within the paper and its supporting information files.
Consent for publication
Ethics approval and consent to participate
NG research is supported by SPM fellowship, CSIR, Government of India. The
study was supported by a grant to KVSR from department of
biotechnologyGovernment of India. The study was also supported by internal grants from
the International center for genetic engineering and Biotechnology (ICGEB),
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1. Agathocleous M , Harris WA . Metabolism in physiological cell proliferation and differentiation . Trends Cell Biol . 2013 ; 23 ( 10 ): 484 - 92 .
2. Jailkhani N , Ravichandran S , Hegde SR , Siddiqui Z , Mande SC , Rao KV . Delineation of key regulatory elements identifies points of vulnerability in the mitogen-activated signaling network . Genome Res . 2011 ; 21 ( 12 ): 2067 - 81 .
3. Bellacosa A , Kumar CC , Di Cristofano A , Testa JR . Activation of AKT kinases in cancer: implications for therapeutic targeting . Adv Cancer Res . 2005 ; 94 : 29 - 86 .
4. Chen J , Somanath PR , Razorenova O , Chen WS , Hay N , Bornstein P , et al. Akt1 regulates pathological angiogenesis, vascular maturation and permeability in vivo . Nat Med . 2005 ; 11 ( 11 ): 1188 - 96 .
5. Cho H , Thorvaldsen JL , Chu Q , Feng F , Birnbaum MJ . Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice . J Biol Chem . 2001 ; 276 ( 42 ): 38349 - 52 .
6. Chen WS , Xu PZ , Gottlob K , Chen ML , Sokol K , Shiyanova T , et al. Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene . Genes Dev . 2001 ; 15 ( 17 ): 2203 - 8 .
7. Tschopp O , Yang ZZ , Brodbeck D , Dummler BA , Hemmings-Mieszczak M , Watanabe T , et al. Essential role of protein kinase B gamma (PKB gamma/ Akt3) in postnatal brain development but not in glucose homeostasis . Development . 2005 ; 132 ( 13 ): 2943 - 54 .
8. Gupta N , Duggal S , Jailkhani N , Chatterjee S , Rao KVS , Kumar A . Dataset to delineate changes in association between Akt1 and its interacting partners as a function of active state of Akt1 protein . Data Brief . 2017 ; 13 : 187 - 91 .
9. Sefton EC , Qiang W , Serna V , Kurita T , Wei JJ , Chakravarti D , et al. MK -2206, an AKT inhibitor, promotes caspase-independent cell death and inhibits leiomyoma growth . Endocrinology . 2013 ; 154 ( 11 ): 4046 - 57 .
10. Ji S , Lin W , Wang L , Ni Z , Jin F , Zha X , et al. Combined Targeting of mTOR and Akt using rapamycin and MK-2206 in the treatment of tuberous sclerosis complex . J Cancer . 2017 ; 8 ( 4 ): 555 - 62 .
11. Nitulescu GM , Margina D , Juzenas P , Peng Q , Olaru OT , Saloustros E , et al. Akt inhibitors in cancer treatment: the long journey from drug discovery to clinical use (Review) . Int J Oncol . 2016 ; 48 ( 3 ): 869 - 85 .
12. Liu P , Cheng H , Roberts TM , Zhao JJ . Targeting the phosphoinositide 3-kinase pathway in cancer . Nat Rev Drug Discov . 2009 ; 8 ( 8 ): 627 - 44 .
13. Trinkle-Mulcahy L , Boulon S , Lam YW , Urcia R , Boisvert FM , Vandermoere F , et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes . J Cell Biol . 2008 ; 183 ( 2 ): 223 - 39 .
14. Kanehisa M , Sato Y , Kawashima M , Furumichi M , Tanabe M. KEGG as a reference resource for gene and protein annotation . Nucleic Acids Res . 2016 ; 44 ( D1 ): D457 - 62 .
15. Chen WS , Peng XD , Wang Y , Xu PZ , Chen ML , Luo Y , et al. Leptin deficiency and beta-cell dysfunction underlie type 2 diabetes in compound Akt knockout mice . Mol Cell Biol . 2009 ; 29 ( 11 ): 3151 - 62 .
16. Arroyo R , Sune G , Zanzoni A , Duran-Frigola M , Alcalde V , Stracker TH , et al. Systematic identification of molecular links between core and candidate genes in breast cancer . J Mol Biol . 2015 ; 427 (6 Pt B): 1436 - 50 .
17. Hirai H , Sootome H , Nakatsuru Y , Miyama K , Taguchi S , Tsujioka K , et al. MK- 2206 , an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo . Mol Cancer Ther . 2010 ; 9 ( 7 ): 1956 - 67 .
18. Pant A , Lee II , Lu Z , Rueda BR , Schink J , Kim JJ . Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin . PLoS ONE . 2012 ; 7 ( 7 ): e41593 .