Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila, but Neither Phosphorylation Is Required for Normal Animal Growth

Abstract

Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt's phosphorylation targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt's growth promoting activity. Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2 activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1 and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.

Full text

Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila,
but Neither Phosphorylation Is Required for Normal
Animal Growth
Sibylle Schleich, Aurelio A. Teleman*
German Cancer Research Center (DKFZ), Heidelberg, Germany
Abstract
Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt’s phosphorylation
targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt’s growth promoting activity.
Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2
activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable
and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif
that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1
and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites
on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the
Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.
Citation: Schleich S, Teleman AA (2009) Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila, but Neither Phosphorylation Is Required for Normal Animal
Growth. PLoS ONE 4(7): e6305. doi:10.1371/journal.pone.0006305
Editor: Andreas Bergmann, University of Texas MD Anderson Cancer Center, United States of America
Received April 27, 2009; Accepted May 18, 2009; Published July 17, 2009
Copyright: ß2009 Schleich, Teleman. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Helmholtz Young Investigator Award. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: a.teleman@dkfz.de
Introduction
The protein complex consisting of Tsc1 (also known as
hamartin) and Tsc2 (also known as tuberin) has emerged in the
past decade as an important regulator of the potent anabolic
kinase TOR complex 1 (TORC1) (for review see [1]). The Tsc1/2
complex appears to sense a large number of inputs such as the
presence of growth factors, cytokines, energy stress and hypoxia,
and integrates this information to regulate the activity of TORC1
via the GTPase Rheb [1]. TORC1 in turn regulates cellular
translation rates to affect both cell growth (and consequently
organismal size) and metabolism [2–4]. This ‘signaling cassette’ is
highly conserved in evolution, and many of the discoveries piecing
together the molecular connections between components of this
cassette were concurrently performed in multiple model systems
such as Drosophila and mice, leading to equivalent results.
One function of the Tsc1/2 complex appears to be to mediate
the activation of TORC1 in response to Akt. The current model
proposes that in response to insulin/IGF signaling, PI3K and
subsequently Akt become activated. Upon activation, Akt
phosphorylates Tsc2 on numerous sites. This inactivates the
Tsc1/Tsc2 complex, relieving the suppression of TORC1 by
Tsc1/2, leading to TORC1 activation and cell growth. This would
provide a molecular link by which insulin-mediated activation of
Akt leads to TORC1 activation, and hence tissue growth.
However, the in vivo relevance of this function for Tsc1/2 is
unclear due to discordant findings in the literature. This model is
supported by a large body of evidence. In both mammalian
systems and in flies, Tsc2 is indeed phosphorylated by Akt in vivo
and in vitro [5–7]. The model predicts that alanine-substitution
mutants of Tsc2 lacking the Akt phosphorylation sites should be
insensitive to Akt activity. Indeed, overexpression of such mutants
leads to a more powerful suppression of TORC1 activity
compared to overexpression of wildtype Tsc2 [5–8], and this
overexpression is able to dominantly block Akt-mediated activa-
tion of TORC1 [5–8]. This is the case in mammalian cell culture,
Drosophila cell culture as well as in Drosophila tissues, and
indicates that at least when Tsc2 is overexpressed, the ability of
Akt to phosphorylate it is functionally relevant. The most rigorous
test, however, to check whether the phosphorylation of Tsc2 by
Akt is functionally important for an animal is to generate mutant
animals in which endogenous Tsc2 is replaced by a non-
phosphorylatable alanine-substitution mutant. This experiment,
asking what happens when Tsc2 cannot be phosphorylated by Akt
in vivo, was performed by Dong and Pan in 2004 [9]. They
generated flies in which they mutated the endogenous Tsc2 gene
and simultaneously expressed either a wildtype Tsc2 or a mutant
Tsc2 in which all four Akt phosphorylation sites were mutated to
alanine or to a phosphomimetic residue. Surprisingly, although
Tsc2 null flies, like mice, die early in development, flies containing
either alanine-substitution or phosphomimicking mutants of Tsc2
were viable, fertile, normally patterned and normal in size and
growth rate [9]. This suggests that at least in Drosophila, although
Akt can and does phosphorylate Tsc2 on multiple sites, this
phosphorylation is functionally not very important.
An open question is how to interpret this result and to reconcile
it with the remaining body of evidence mentioned above. Is
phosphorylation of Tsc2 by Akt important for Akt to drive tissue
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growth in vivo or not? One option is that the result by Dong and
Pan reflects something specific to Drosophila. Indeed, as was noted
previously [7], Drosophila Tsc1 - the binding partner of Tsc2 -
also contains a consensus Akt phosphorylation site (Ser533) which
is not conserved in mammals. Both Tsc1 and Tsc2 need to be
active to achieve normal activity of the complex, and recently it
has been shown that phosphorylation of Tsc1 (e.g. by IKKb, [10])
can inhibit Tsc1/2 complex activity in cell culture. Thus it is
possible that Akt phosphorylates both partners of the Tsc1/2
complex in Drosophila, and that unless phosphorylation of both
partners is simultaneously abrogated, Akt will be able to disrupt
Tsc1/2 function. This possibility is strengthened by the fact that
Ser533 is reported to be phosphorylated in vivo in Drosophila
KC167 cells, detected by mass spectroscopy (www.phosphopep.
org [11]).
In this study, we examine whether Tsc1 is phosphorylated by
Akt in Drosophila, and the physiological consequences of this
phosphorylation. We provide evidence that Akt phosphorylates
Tsc1 at Ser533 and that this phosphorylation is induced by insulin
signaling. We test genetically the requirement for this phosphor-
ylation by engineering Tsc1 mutants in which the Akt phosphor-
ylation sites are mutated to nonphosphorylatable (Tsc1
S533A
)or
phosphomimetic (Tsc1
S533D
) residues, and show that in both cases
the flies are rescued to full viability and size. To ask whether the
phosphorylation of dTsc1 and dTsc2 by Akt are functionally
redundant, we genetically engineer flies in which both Tsc1 and
Tsc2 are simultaneously replaced with mutant versions that cannot
be phosphorylated by Akt (Tsc1
S533A
, Tsc2
T437A/S924A/T1054A/
T1518A
). Surprisingly, these animals are also viable and normal in
size and growth rate. This shows that phosphorylation of both
Tsc1 and Tsc2 is not required for Akt to drive tissue growth in
Drosophila, indicating that other targets of Akt must be
responsible for Akt’s growth-promoting activity. We do find,
nonetheless, that these animals have mild metabolic defects,
raising the possibility that the regulation of Tsc1/2 by Akt plays a
fine-tuning role in organismal metabolism. This would be similar
to what is seen with other components of the pathway, such as
Rictor and Melted, which play important yet modulatory
functions during animal development [12,13].
Results
Akt phosphorylates dTsc1 on Ser533
As was previously noted [7], Drosophila Tsc1 contains a perfect
Akt phosphorylation consensus (R-x-R-x-x-S/T) at Ser533 –
RNRMAS – which is not conserved in mammalian Tsc1 proteins.
This raises the possibility that Akt phosphorylates dTsc1 in
addition to dTsc2 in Drosophila. Furthermore, using a proteomic
approach in which phosphopeptides were identified from extracts
of Drosophila Kc167 cells using mass spectroscopy, Aebersold and
colleagues have detected phosphorylation on Ser533 of endoge-
nous Tsc1, indicating that this site is phosphorylated in vivo by an
unknown kinase (www.phosphopep.org [11]). Therefore, we
decided to test if Akt phosphorylates dTsc1 at Ser533. To detect
phosphorylation at this site, we transfected S2 cells with a
construct expressing myc-tagged Tsc1. We then immunoprecip-
itated Tsc1 using the myc tag, and detected phosphorylation using
the Phospho-(Ser/Thr) Akt Substrate antibody from Cell Signal-
ing which recognizes (R-x-R-x-x-phosphoS/T) epitopes. This
antibody should recognize Ser533 on Tsc1 if it is phosphorylated.
When S2 cells were transfected to express wildtype Tsc1, a weak
phospho-signal could be detected in the myc-Tsc1 immunopre-
cipitate (Figure 1A, Lane 1) which increased progressively in
strength when the cells were treated with insulin for 20, 40 or
60 minutes prior to lysis (Figure 1A, Lanes 2–4), indicating that
this phosphorylation is insulin responsive. We then tested which
site on Tsc1 is responsible for this signal. When S2 cells were
transfected to express wildtype myc-Tsc1, the signal in the anti-
myc immunoprecipitate increased in strength upon insulin
treatment, consistent with the previous result (Figure 1B, lanes 3
and 4). This signal was not detectable if cells were not transfected,
indicating the phospho-specific antibody is specifically detecting
myc-Tsc1 in the myc-IP (Figure 1A, lanes 1 and 2). Furthermore,
no phospho-specific signal was detectable if we transfected a myc-
tagged Tsc1 in which Ser533 was mutated to alanine, indicating
that the phospho-specific antibody is specifically recognizing
phosphorylation on Ser533 (Figure 1, lanes 5 and 6). Together,
these data suggested that Tsc1 is phosphorylated on Ser533 by a
kinase that is activated in response to insulin signaling. Since
Ser533 lies within an Akt phosphorylation motif, and since Akt is
activated upon insulin stimulation, a likely candidate for this kinase
is Akt.
To ask whether the increased phosphorylation of Ser533 on
dTsc1 in response to insulin is due to Akt, we tested whether
knockdown of Akt was able to blunt this response. We transfected
S2 cells with myc-Tsc1(WT), treated the cells with dsRNA against
Akt or a control, and then treated the cells with or without insulin
before detecting phosphorylation on Tsc1. As shown in Figure 1C,
insulin treatment caused an increase in Tsc1 phosphorylation in
control cells (lanes 1 and 2), but not in cells treated with Akt
dsRNA (lanes 3 and 4). As observed also by others [13], the
Phospho-(Ser/Thr) Akt Substrate antibody from Cell Signaling
shows a background signal (both top and bottom panel, Fig. 1C).
This background signal, most clearly visualized when probing total
cell lysates (bottom panel, Figure 1C) is retained when Akt is
completely knocked-down, as controlled with anti-Akt antibody
and by loss of S6K phosphorylation (Fig. 1C). Since the banding
pattern visible when cell lysates are probed with this antibody are
similar in the presence and absence of Akt (bottom panel Fig. 1C),
this likely reflects residual binding of the antibody to non-
phosphorylated R-x-R-x-x-S/T motifs. This has also been
reported by others (Figure 1G in [13]). Despite the background
signal, the fact that the phospho-signal on Tsc1 no longer increases
upon insulin treatment when Akt is removed, indicates Akt is
responsible for the insulin-induced phosphorylation of dTsc1 at
Ser533.
dTsc1 phosphorylation by Akt is dispensable in vivo in
Drosophila
Some groups have reported that binding between dTsc1 and
dTsc2 depends on phosphorylation of Tsc2 [7], whereas others
have reported that it does not [9]. We also could not detect any
changes in dTsc1/dTsc2 binding in the presence or absence of
insulin (not shown), so we could not use a binding assay to probe
the effect of Tsc1 phosphorylation on Tsc1/2 function. Therefore
we decided to move to an in vivo model.
To test the physiological relevance of this phosphorylation event
in vivo, we genetically engineered flies in which endogenous Tsc1
was replaced with various mutant versions. To achieve this, we
generated transgenic flies ubiquitously expressing either wildtype
Tsc1 (Tsc1
WT
), or Tsc1 variants where Ser533 was mutated to
non-phosphorylatable alanine (Tsc1
S533A
) or to a phosphomimick-
ing residue (Tsc1
S533D
). These transgenes were then crossed into a
Tsc1
29
mutant background, in a manner similar to that done by
Dong and Pan for Tsc2 [9]. The Tsc1
29
mutation replaces amino
acid 61 with a stop codon, truncating most of the protein, leading
to a predicted null [14]. While Tsc1
29
mutant animals die very
early around the embryo-larval transition [14], presence of the
dAkt Phosphorylates dTsc1
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Tsc1
WT
transgene was able to rescue them to adulthood,
generating a viable stock with no obvious defects. By picking first
instar larvae and seeding them at fixed density on standard flyfood,
we found that 83% of control w
1118
larvae survived to adulthood,
and 69% of Tsc1
29
mutants were rescued to adulthood with the
Tsc1
WT
transgene (Figure 2B, ‘‘WT’’). We then chose transgenes
expressing Tsc1
S533A
or Tsc1
S533D
at levels similar to Tsc1
WT
(Figure 2A, lanes 2, 4 and 5), introduced them into the Tsc1
29
background, and found that they were also able to rescue the
mutant flies as efficiently as the Tsc1
WT
construct: 61% and 63%
of Tsc1
29
mutants were rescued to adulthood with the Tsc1
S533A
and Tsc1
S533D
transgenes respectively (Figure 2B). Furthermore,
flies rescued by the wildtype and two mutant constructs showed
similar developmental timing, gauged by pupation curves
(Figure 2C), and similar final animal size, measured by wing area
(Figure 2D). Although wing size was mildly reduced in both
Tsc1
S533A
and Tsc1
S533D
flies compared to Tsc1
WT
flies, opposite
effects would be expected from the alanine-substitution and
phosphomimicking transgenes, making it unclear if this mild
reduction is of significance. In sum, the ability of all three
transgenes to rescue Tsc1
29
mutants from early lethality to
adulthood suggests that phosphorylation of Tsc1 by Akt is not
critical for normal development in Drosophila.
Flies simultaneously lacking Akt phosphorylation of Tsc1
and Tsc2 are viable but have mild metabolic defects
The Tsc1 and Tsc2 proteins work together as a complex to
achieve maximal activity, and recent reports indicate that
phosphorylation of either Tsc1 or Tsc2 can lead to regulation of
the complex in cell culture [1,10]. To test whether phosphorylation
by Akt of Tsc1 and Tsc2 might be acting redundantly, we generated
flies in which both endogenous Tsc1 and Tsc2 were simultaneously
replaced with alanine-substitution mutants (tsc1
29
, gig
192
,
Tsc1
S533A
,Tsc2
T437A/S924A/T1054A/T1518A
)(gig
192
; Tsc2
T437A/
S924A/T1054A/T1518A
flies kindly provided by D. Pan [9]). To our
surprise, these animals were also viable (Figure 2B, ‘‘double’’), and
had similar growth rates and final size compared to animals
harboring the Tsc1
WT
transgene (Figures 2C and 2D, ‘‘double’’).
This is in stark contrast to animals lacking Tsc1, Tsc2, Akt, or Rheb,
all of which are lethal early in development [14–18]. This indicates
that even if the ability of Akt to phosphorylate both partners of the
Tsc1/2 complex is abrogated, flies are quite normal in terms of
growth, and that in Drosophila the ability of Akt to drive tissue
growth does not depend strongly on the Tsc1/2 complex.
Both insulin signaling and TORC1 are known to regulate
animal metabolism in addition to growth. Indeed, flies mutant for
a number of components of the pathway, such as rictor or melted,
display very mild growth impairments, but have strong metabolic
defects [12,13,19]. This suggests that animal metabolism is more
sensitive to TOR activity than animal growth. Therefore, in order
Figure 1. Drosophila Tsc1 is phosphorylated by Akt on Ser533.
(A) Phosphorylation of Tsc1 increases with insulin treatment. S2 cells
transfected with constructs to express myc-Tsc1 and His-Tsc2 were
treated without insulin (0 min) or with insulin (10 mg/mL) for indicated
times (20, 40 or 60 min). Cells were then lysed and myc-Tsc1
immunoprecipitated using anti-myc antibody. Immunoprecipitates
were probed with anti-myc antibody as a loading control, and anti-
Phospho-(Ser/Thr) Akt Substrate antibody to detect phosphorylation of
Tsc1. (Ser533 is part of an Akt phosphorylation consensus motif). (B)
Tsc1 is phosphorylated on Ser533 in response to insulin treatment.
Untransfected S2 cells (-) or S2 cells transfected with constructs to
express either myc-Tsc1
WT
(‘‘WT’’) or myc-Tsc1
S533A
(‘‘S533A’’) together
with His-Tsc2 were treated with or without insulin (10 mg/mL) for
1 hour prior to lysis and immunoprecipitation with anti-myc antibody.
Immunoprecipitates were probed with anti-myc antibody as a loading
control, and anti-Phospho-(Ser/Thr) Akt Substrate antibody to detect
phosphorylation of Tsc1. (C) Knockdown of Akt abrogates the increase
in phosphorylation of Tsc1 on Ser533 induced by insulin treatment. S2
cells transfected with expression constructs for myc-Tsc1
WT
and His-
Tsc2 were treated with control dsRNA or Akt dsRNA for 4 days prior to
insulin treatment (10 mg/mL for 1 hour), lysis and anti-myc immuno-
precipitation. Immunoprecipitates were probed with anti-myc as a
control and anti Phospho-(Ser/Thr) Akt Substrate antibody to detect
phosphorylation of Tsc1 Ser533. Despite efficient knockdown of Akt
(seen by lack of Akt protein and S6K phosphorylation in lanes 3 and 4),
anti Phospho-(Ser/Thr) Akt Substrate antibody displays background
binding in total cell lysates, as previously reported also by others.
doi:10.1371/journal.pone.0006305.g001
dAkt Phosphorylates dTsc1
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to not overlook more mild defects, we tested whether phosphor-
ylation of the Tsc1/2 complex by Akt might affect organismal
metabolism by measuring animal lipid levels. Although animals in
which endogenous Tsc1 was replaced with either Tsc1
S533A
or
Tsc1
S533D
did not reproducibly show alterations in lipid levels
(Figure 3), animals in which both Tsc1 and Tsc2 were
simultaneously replaced with alanine-substitution mutants were
mildly leaner than controls (Figure 3, ‘‘double’’ vs ‘‘WT’’,
ttest = 0.01). This suggests that phosphorylation of the Tsc1/2
complex by Akt might possibly be involved in the more subtle
regulation of animal metabolism, as is seen with other modulators
of the pathway such as Rictor or Melted [12,13,19].
Discussion
dAkt phosphorylates dTsc1
We present evidence here that Drosophila Tsc1 is phosphor-
ylated on Ser533 by Akt. Although this serine is conserved in
mouse and human Tsc1, the R-x-R-x-x-S motif is not conserved.
(In human Tsc1 the respective sequence is 519-THSAAS-524).
Since Akt does not absolutely require the full R-x-R-x-x-S motif to
recognize its targets [20], we tested whether human Tsc1 is also
phosphorylated on Ser524, but could not detect any phosphory-
lation by mass spectroscopy with immunopurified hTsc1 from
HEK293 cells (data not shown). Therefore, we believe this is likely
a Drosophila-specific phosphorylation. This phosphorylation site is
in very close proximity to Ser487 and Ser511 of human Tsc1,
which were recently shown to be phosphorylated by IKKb[10],
leading to regulation of Tsc1/2 function in cell culture. Therefore,
it is possible that this clustering of phosphorylation sites in one
region of Tsc1 is of functional significance, in particular since it is
close to the domain that interacts with Tsc2.
Is phosphorylation of the Tsc1/2 complex by Akt
important?
The finding by Dong and Pan, that flies lacking Akt
phosphorylation sites on Tsc2 are viable and normal in size, was
Figure 2. Flies lacking Akt phosphorylation sites on Tsc1 and Tsc2 are viable and normal in size. (A) Expression levels of myc-Tsc1 in fly lines
homozygous for the Tsc1
29
mutation, rescued by ubiquitous expression of Tsc1
WT
, Tsc1
S533A
or Tsc1
S533D
, or flies homozygous for both the Tsc1
29
and
Tsc2
192
mutations rescued to viability by ubiquitous expression of both Tsc1
S533A
and Tsc2
T437A/S924A/T1054A/T1518A
(‘‘Tsc1
S533A
,Tsc2
4A
’’). (B,C,D) Survival
rates (B), pupation curves (C) and relative adult wing sizes (D) of animals seeded as L1 larvae under controlled growth conditions for genotypes w
1118
(‘‘w
1118
’’), Tsc1
29
homozygotes rescued by ubiquitous expression of Tsc1
WT
(‘‘WT’’), Tsc1
S533A
(‘‘S533A’’) or Tsc1
S533D
(‘‘S533D’’), or flies homozygous for
both the Tsc1
29
and Tsc2
192
mutations rescued to viability by ubiquitous expression of both Tsc1
S533A
and Tsc2
T437A/S924A/T1054A/T1518A
(‘‘double’’).
doi:10.1371/journal.pone.0006305.g002
dAkt Phosphorylates dTsc1
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surprising [9]. Since we found here that Akt also phosphorylates
Tsc1 in Drosophila, this raised the possibility that the phosphor-
ylation of Tsc1 and Tsc2 by Akt are functionally redundant, and
that a phenotype is only revealed when both are abrogated.
However, to our surprise, we found that flies simultaneously
lacking Akt phosphorylation sites on both Tsc1 and Tsc2 are also
viable and almost normal in size, reinforcing the conclusion that
the connection from Akt to TOR via the Tsc1/2 complex is not
critical for normal size and growth. Since Akt strongly activates
TORC1 activity and induces tissue growth, this suggests other
targets of Akt must be responsible for these effects. Recently,
PRAS40 has also been suggested to link Akt to TOR: some groups
have reported that Akt can phosphorylate PRAS40, thereby
relieving the inhibition of TOR by PRAS40 [21,22]. Although
other groups have reported conflicting data, or alternate
interpretations of this data [23–25], it is possible that Akt activates
TOR via both Tsc1/2 and PRAS40 in a redundant manner, or
that other unknown links between Akt and TOR exist. This
redundancy would generate a more ‘robust’ system in which
TORC1 activity is held in check by two independent pathways,
both of which are downstream of Akt. Furthermore, a number of
inputs regulate activity of the Tsc1/2 complex, phosphorylation by
Akt being only one of them.
One interpretation of our data is that abrogation of the ability of
Akt to phosphorylate the Tsc1/2 complex has no functional
consequences whatsoever for the animal. Since we find this hard to
believe, we tested whether there might be more mild defects in the
mutant flies. TOR regulates both tissue growth and organismal
metabolism. Some mutations in the fly with mild effects on TOR
activity cause small or negligible alterations in animal size, but
significant alterations in metabolic parameters such as total body
lipid levels [12,13]. This suggests that metabolic regulation is more
sensitive to TOR activity than animal size. Therefore, we tested
whether flies simultaneously lacking Akt phosphorylation sites on
Tsc1 and Tsc2 are metabolically normal. Indeed, we found that
these flies have a mild reduction in body lipid levels. Therefore it is
possible that the link between Akt and TOR via the Tsc1/2
complex is more important for fine-tuning animal metabolism
than for controlling animal growth.
Materials and Methods
Molecular Biology & Fly stocks
Vectors for expressing myc-tagged dTsc1 and His/V5-tagged
Tsc2 under control of the actin promoter were a kind gift from
Duojia Pan [14]. Point mutations in TSC1 at serine 533 were
introduced by PCR to obtain an alanine mutant (oligo
GAACCATTTCCACTGTAggcTGCCATACGATTGCG), or
an aspartic acid mutant (oligo GAACCATTTCCACTG-
TAgtcTGCCATACGATTGCG) Final constructs were rese-
quenced to confirm presence of the mutations. To generate
transgenic flies, Tsc1-WT, Tsc1-S533A and Tsc1-S533D were
subcloned into a pCasper4-based vector containing a tubulin
promotor and an SV40 polyA. Flies containing the gigas
192
mutation, expressing either wildtype Tsc2 or Tsc2
T437A/S924A/
T1054A/T1518A
were a kind gift from Duojia Pan [9].
Cell culture, Immunoprecipitations and Antibodies
Transfections of Tsc1 and Tsc2 constructs were carried out in S2
cells grown in SFM medium using Cellfectin Reagent (Invitrogen).
Twenty hours after transfection, cells were treated with or without
bovine insulin for one hour (10 mg/mL, Sigma), then lysed in lysis
buffer (50 mM Tris-pH 7.5, 150 mM NaCl
2
, 1% Triton X-100),
containing protease and phosphatase inhibitors (Roche). Immuno-
precipitations were performed using rabbit anti myc antibody from
Cell Signaling (#71D10), and Protein-A agarose beads (Roche).
dsRNA targeting both AKT isoforms was generated using oligos
containing T7 promoter sequences fused to GAGATT-
GTGTGTGTTTCGT or GTTCCGGAATCGTGTGTA. dsRNA
was added to the medium at 12 mg/mL for 4 days.
Antibodies: anti p-Thr398 dS6k (Cell Signaling, #9209), anti-
AKT (Cell Signaling #9272), anti myc (Dianova MA1-980), anti-
tubulin (DS Hybridoma Bank AA4.3-s), anti dS6K (kind gift from
Mary Stewart).
Fat measurement
Age and nutrient controlled flies were collected three or five
days after hatching and subjected to fat measurement as previously
described [19]. Samples of at least five flies were homogenized in
ice-cold homogenization buffer (0.05% Tween 20 in H2O). A
sample was kept for Bradford protein measurement and assayed
immediately. The remaining homogenate was heat-inactivated at
70uC for 5 min. After addition of lipoprotein lipase (Sigma 62333,
final concentration 0.25 mg/ml, 37 degrees, overnight) glycerol
from the reaction was quantified using Free Glycerol Reagent
(Sigma F6428). Experiments were done in quintuplicate.
Acknowledgments
Anti-tubulin antibody was obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD and
maintained by The University of Iowa. We thank DJ Pan for providing us
with Drosophila strains and S Cohen for comments on the manuscript.
Author Contributions
Conceived and designed the experiments: AAT. Performed the experi-
ments: SS AAT. Analyzed the data: SS AAT. Contributed reagents/
materials/analysis tools: AAT. Wrote the paper: SS AAT.
Figure 3. Flies lacking Akt phosphorylation sites on both Tsc1
and Tsc2 are slightly lean. Triglyceride levels normalized to total
body protein for Tsc1
29
homozygotes rescued by ubiquitous expression
of Tsc1
WT
(‘‘WT’’), Tsc1
S533A
(‘‘S533A’’) or Tsc1
S533D
(‘‘S533D’’), or flies
homozygous for both the Tsc1
29
and Tsc2
192
mutations rescued to
viability by expression of both Tsc1
S533A
and Tsc2
T437A/S924A/T1054A/T1518A
(‘‘double’’). * indicates statistical significance (ttest = 0.01).
doi:10.1371/journal.pone.0006305.g003
dAkt Phosphorylates dTsc1
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dAkt Phosphorylates dTsc1
PLoS ONE | www.plosone.org 6 July 2009 | Volume 4 | Issue 7 | e6305