Phosphatidylinositol 3,4,5-trisphosphate modulation
in SHIP2-deficient mouse embryonic fibroblasts
Daniel Blero
1
, Jing Zhang
1
, Xavier Pesesse
1
, Bernard Payrastre
2
, Jacques E. Dumont
1
,
Ste
´
phane Schurmans
3
and Christophe Erneux
1
1 Interdisciplinary Research Institute (IRIBHM), Universite
´
Libre de Bruxelles, Belgium
2 INSERM U563, Departement d’Oncogenese et Signalization dans les Cellules Hematopoietiques, Ho
ˆ
pital Purpan, Toulouse Cedex, France
3 IRIBHM, IBMM, Gosselies Belgique
The SHIPs (SH2 domain containing inositol 5-phos-
phatases) are members of the inositol 5-phosphatase
family. Two isoenzymes, named SHIP1 and SHIP2
have been identified and characterized [1–4]. The cellu-
lar and tissue distribution of SHIP2 is very wide [5],

SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase,
includes a series of protein interacting domains and has the ability to
dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P
3
]
in vitro. The present study, which was undertaken to evaluate the impact of
SHIP2 on PtdIns(3,4,5)P
3
levels, was performed in a mouse embryonic
fibroblast (MEF) model using SHIP2 deficient (– ⁄ –) MEF cells derived
from knockout mice. PtdIns(3,4,5)P
3
was upregulated in serum stimulated
– ⁄ – MEF cells as compared to + ⁄ + MEF cells. Although the absence of
SHIP2 had no effect on basal PtdIns(3,4,5)P
3
levels, we show here that
this lipid was significantly upregulated in SHIP2 – ⁄ – cells but only after
short-term (i.e. 5–10 min) incubation with serum. The difference in
PtdIns(3,4,5)P
3
levels in heterozygous fibroblast cells was intermediate
between the + ⁄ + and the – ⁄ – cells. In our model, insulin-like growth
factor-1 stimulation did not show this upregulation. Serum stimulated
phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable
between + ⁄ + and – ⁄ – cells. Moreover, protein kinase B, but not mitogen
activated protein kinase activity, was also potentiated in SHIP2 deficient
cells stimulated by serum. The upregulation of protein kinase B activity
in serum stimulated cells was totally reversed in the presence of the
PI 3-kinase inhibitor LY-294002, in both + ⁄ + and – ⁄ – cells. Altogether,

mulation, SHIP2 is translocated to the plasma mem-
brane, where it inhibits the insulin-specific subcellular
redistribution of Akt2 [12]. The expression of SHIP2
was enhanced in an animal model of type 2 diabetes
which was accompanied by an attenuation of insulin
signalling [13]. However, a role for SHIP2 has also
been suggested in other pathways: in rat vascular
smooth muscle cells, SHIP2 downregulates PDGF and
IGF-1 mediated signalling downstream of PI 3-kinase
[14]. In glioblastoma cells, SHIP2 inhibits protein kin-
ase B (PKB) and provokes a potent cell cycle arrest in
G
1
[15]. SHIP2 could play an essential role in cell
adhesion and spreading as shown in HeLa cells [16]. A
regulatory role for SHIP2 in M-CSF-induced signalling
has been recently suggested [8]. SHIP2 also functions
in the maintenance and dynamic remodelling of actin
structures as well as in endocytosis and downregula-
tion of the EGF receptor [17].
In vivo, homozygous disruption of SHIP2 by remo-
ving exons 19–29 causes severe hypoglycemia and
death within a few hours after birth. Heterozygous dis-
ruption of this gene leads to hypersensitivity to insulin
demonstrated by the increased glycogen synthesis in
skeletal muscles in response to insulin. Injection of
d-glucose resulted in a more rapid glucose clearance
in SHIP2+ ⁄ – than in SHIP2+ ⁄ + mice. Moreover,
the incidences of spontaneous or irradiated-induced
tumours were not affected in SHIP2+ ⁄ – mice [18].

activities were also decreased in SHIP2 transfected cells
suggesting that SHIP2 is a down-regulator of both arms
of receptor tyrosine kinase activation [10,15,22,23]. The
present study was therefore undertaken to establish the
extent of PtdIns(3,4,5)P
3
regulation in SHIP2 – ⁄ – cells
derived from MEF cells. Although the absence of
SHIP2 had no effect on basal PtdIns(3,4,5)P
3
levels, we
show here that this lipid was significantly upregulated
in SHIP2 – ⁄ – MEF cells but only after short-term
(i.e. 5–10 min) incubation with serum. In our model,
IGF-1 stimulation did not show this upregulation
and PtdIns(4,5)P
2
levels were comparable between
SHIP2+ ⁄ + and – ⁄ – MEF cells.
Results
Status of SHIP2, PTEN, insulin and IGF-1 receptor
expression in SHIP2 +/+ and –/– MEF cells
SHIP2 – ⁄ – mice were obtained as reported previously
[18]. As our SHIP2 – ⁄ – mice died very shortly after
birth, we chose to work with MEF cells as a model
to measure the 3-phosphorylated phosphoinositides.
MEF cells were prepared from embryos of hetero-
zygous crosses and genotyped by PCR analysis. Two
series of MEF cells (1 and 2) were prepared from
two independent crosses to validate the measurements

[15], we compared the levels of [
3
H]PtdIns(4,5)P
2
and
[
3
H] phosphatidylinositol 4-phosphate (PtdIns4P) after
labelling the cells with [
3
H]inositol in the presence of
10% FBS for 72 h: the amount of [
3
H]PtdIns(4,5)P
2
and [
3
H]PtdIns4P did not change significantly between
+ ⁄ + and – ⁄ – MEF cells (Fig. 2A). Similar results
were obtained when we labelled the cells with
[
32
P]orthophosphate for more than 4 h. In our assay
SHIP2
MEF SHIP2
+/+
1
MEF SHIP2
+/+
2

MEF SHIP2
-
/
-
1
MEF
SHIP2
+/+
1
MEF SHIP2
-
/
-
2
CHO
-
IR
Fig. 1. Western blot analysis of MEF
SHIP2 + ⁄ +, + ⁄ –and–⁄ – cells. Twenty
micrograms of proteins from a lysate made
of MEF cells or CHO-IR were applied to
SDS gels. Immunodetection was performed
with antibodies against SHIP2, PTEN, IGF-1
and the insulin receptor (IGF-1R and InsR).
MEF cells 1 and 2 were from two
independent preparations of cells.
PtdIns(3,4,5)P
3
levelsLabelling with [
3

0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
0 min 10 min
%(of PtdIns(4,5)P2)
SHIP2+/+
SHIP2-/-
PtdIns(3,4,5)P
3
levels
0
0,2
0,4
0,6
0,8
1
1,2
1,4
0 min 10 min
%(of PtdIns(4,5)P2)
SHIP2+/+
SHIP2+/-
SHIP2-/-
Fig. 2. PtdIns(3,4,5)P

P] and stimulated for 10 min by 10% FBS. The data are means of
duplicates ± SD. The data are representative of two different experiments.
Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts D. Blero et al.
2514 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS
of [
32
P]-labelled lipids, we scraped off together the
region of the TLC containing both [
32
P]PtdIns(3,4,5)P
3
and [
32
P]PtdIns(4,5)P
2
, the levels of [
32
P]-labelled
3-phosphoinositides were normalized with respect to
[
32
P]PtdIns(4,5)P
2
.
SHIP2 modulated PtdIns(3,4,5)P
3
levels after
short-term serum stimulation
The levels of PtdIns(3,4,5)P
3

PtdIns(3,4,5)P
3
were not different between + ⁄ + and
– ⁄ – cells (Fig. 2D).
In contrast to serum, IGF-1 stimulation resulted in
maximal production of PtdIns(3,4,5)P
3
after 2 min and
no significant differences in PtdIns(3,4,5)P
3
levels were
seen between SHIP2+ ⁄ +and – ⁄ – cells (Fig. 3B).
When the MEF cells were stimulated with insulin
(1–100 nm), we did not see any significant increase in
PtdIns(3,4,5)P
3
levels in contrast to CHO-IR cells sti-
mulated with insulin that were used as positive control
(data not shown).
SHIP2 did not modulate PtdIns(3,4,5)P
3
levels
after long-term serum stimulation
Previous studies in MEF cells deficient in PTEN have
shown that PtdIns(3,4,5)P
3
was upregulated about
twofold in – ⁄ – cells after 4 h of incubation in the
presence of 5% FBS. The data suggested that
PtdIns(3,4,5)P

0,2
0,4
0,6
0,8
1
1,2
0510
TIME (minute)
0510
TIME (minute)
%(of PtdIns(4,5)P
2
)
%(of PtdIns(4,5)P
2
)
SHIP2+/+
SHIP2-/-
A
PtdIns(3,4,5)P
3
levels
PtdIns(3,4,5)P
3
levels
+serum
0
0,1
0,2
0,3

3
, i.e. 5 min for FBS and 2 min for IGF-1.
Therefore, changes in PI 3-kinase activity are probably
not responsible for the upregulation of PtdIns(3,4,5)P
3
levels in SHIP2 – ⁄ – MEF cells.
Effect of serum on PKB and MAP kinase activities
In overexpression studies, SHIP2 causes PKB inactiva-
tion and MAP kinase inhibition [10,15,22,23]. Activa-
ted PKB was detected using phosphospecific antibodies
against T308 and S473. PKB activity was upregulated
in serum stimulated SHIP2 –⁄ – cells as compared to
+ ⁄ + cells (Fig. 6A). A similar result was obtained by
using an enzymatic assay for PKB after immunopre-
cipitation of PKB. The net increase in PKB activity in
serum stimulated cells was approximately two times
higher in SHIP2 – ⁄ – cells as compared to + ⁄ + cells
(Fig. 6B). We also showed that the upregulation of
PKB phosphorylation was totally reversed when cells
were preincubated in the presence of the PI 3-kinase
inhibitor LY-294002 (Fig. 7). In contrast, MAK kinase
activities (p-Erk1 ⁄ 2) following serum stimulation were
not different between the two types of cells (Fig. 6A).
Stimulation of MEF with various agonists
MEF cells were also stimulated for 5 min by various
agonists: EGF, hepatocyte growth factor (HGF), b
fibroblast growth factor (FGF), IGF-1 and PDGF
(Fig. 8). HGF, b FGF, IGF-1 (1 nm), did not increase
PtdIns(3,4,5)P
3

Origin
PI3P
FBS10%
FBS10% +LY
B
+/+
-/-
+/+
-/-
Origin
PI3P
SHIP2 MEF
IGF-1 10 nM IGF-1 10 nM +LY
C
-
LY + LY
Fig. 5. PI 3-kinase activity in serum or IGF-1
stimulated SHIP2 + ⁄ +and–⁄ – MEF cells
PI 3-kinase activty was measured in (A) con-
trol, and in stimulated cells (B) 10% serum
for 5 min and (C) 10 n
M IGF-1 for 2 min.
The assay was performed as described.
When used, LY-294002 (25 l
M) was added
to the preincubation for 30 min.
PtdIns(3,4,5)P
3
levels
0

cells (Fig. 8). In order to verify that the agonists used
stimulated MEF cells efficiently, we determined MAP
kinase activity (p-Erk1 ⁄ 2) in the two types of cells: the
agonists tested also stimulated phospho MAP kinase
activity in both SHIP2 + ⁄ + and – ⁄ – cells (Fig. 9).
Discussion
SHIP2 is a typical signalling enzyme potentially
involved in the biochemical cascade of many growth
factors and insulin [2,4,7,10–15,23]. Its sequence shows
the presence of an SH2 domain, proline rich sequences,
a NPXY site that can be phosphorylated on tyrosine
and a catalytic domain which is typical for a member
of the inositol 5-phosphatase family. SHIP2 appears to
be able to dephosphorylate at the 5-position of the
inositol ring of PtdIns(4,5)P
2
, PtdIns(3,4,5)P
3
and ino-
sitol tetrakisphoshate in vitro [7,15,21].
SHIP2
p-ser
473
PKB
A
B
p-thr
308
PKB
total PKB

cates ± SD.
SHIP2 MEF
+/+
-/-
+/+
-/-
+/+
-/-
+/+
-/-
LY 294002
FBS 10%
FBS 10% + LY294002
Control
+/+
-/-
+/+
-/-
+/+
-/-
+/+
-/-
P-Ser
473
PKB
Total PKB
Fig. 7. Phospho PKB activity in SHIP2 + ⁄ +and–⁄ – cells MEF cells.
MEF cells were stimulated in the 10% serum for 5 min in the pres-
ence and absence of LY-294002 (25 l
M). Protein (20 lg) was

30n
g
/ml
%(of tP dI sn4(,5P)2)
SHIP2+/+
SHIP2-/-
0
0,2
0,4
0,6
0,8
1
1,2
S
N
BF
S
%01

G
E
F
5
0
g
n
m
/
l
GH

1
n0
M
P
D
FG
3
n0
g
/
lm
SHIP2+/+
SHIP2-/-
PtdIns(3,4,5)P
3
levels ( 5 min stimulation)
%(of tP dI sn4(5,P)2)
Fig. 8. PtdIns(3,4,5)P
3
and PtdIns(3,4)P
2
levels in SHIP2 + ⁄ +and
– ⁄ – MEF cells. + ⁄ + and – ⁄ – MEF cells were stimulated by 10%
FBS, EGF 50 ngÆmL
)1
, HGF 15 ngÆmL
)1
, b FGF 100 ngÆmL
)1
, IGF-1

not known whether the phenotype was influenced by
this second gene deletion. The second knockout mice,
in which the first 18 exons were deleted, showed a dif-
ferent phenotype with normal insulin and glucose tol-
erances; the mice were however, resistant to dietary
obesity [19]. Interestingly, an increased activation of
PKB phosphorylation was observed in skeletal muscle
and liver of these SHIP2 null mice on stimulation with
insulin [19]. The data obtained in analysing the pheno-
types of both knockout mice are consistent with
SHIP2 being directly responsible for the dephosphory-
lation of PtdIns(3,4,5)P
3
. This is not to say that SHIP2
is only acting as a phosphoinositide 5-phosphatase
(EC 3.1.3.36). For example, the SHIP2 C-terminal
region is quite specific as compared to that of SHIP1
and could interact with specific protein partners such
as filamin or c-Cbl associated protein thereby provi-
ding multiple molecular interactions and possible bio-
chemical regulation mechanisms of its activity and
localization [30,31]. The presence of SHIP2 in mem-
brane ruffles has been reported and this could account
for the regulation of actin rearrangement by regulating
local levels of SHIP2 lipid substrate and ⁄ or interacting
with cytoskeleton regulatory proteins [30]. The trans-
location of SHIP2 to plasma membranes upon insulin
stimulation and the requirement for the negative regu-
lation of insulin signalling could account for SHIP2
specificity [32]. The role of tyrosine phosphorylation of

kinase activity) was potentiated in serum stimulated
SHIP2 – ⁄ – cells with this effect being completely
reversed in the presence of LY-294002. Serum stimu-
lated PI 3-kinase activity appeared to be comparable
between SHIP2 + ⁄ + and – ⁄ – cells and in both
cases, the activity was decreased in the presence of
LY-294002. Therefore, the results obtained with
PtdIns(3,4,5)P
3
in our study could not be explained
by an upregulation of PI 3-kinase in serum stimulated
SHIP2 – ⁄ – cells. We concluded that the increase in
PtdIns(3,4,5)P
3
levels and PKB activity measured in
our study is a consequence of an effect on
PtdIns(3,4,5)P
3
dephosphorylation. Stambolic et al.
[26] reported that PtdIns(3,4,5)P
3
levels were also
potentiated (about twofold) in PTEN – ⁄ – cells that
had been incubated and labelled with 5% FBS for
4 h; however, no kinetics were provided for compari-
son with our data. In contrast, in our study no
change in PtdIns(3,4,5)P
3
levels were seen in SHIP2
depleted cells as compared to + ⁄ + cells either at the

IGF 1 10nM
PDGF 30ng/ml
FBS 10%
control
Erk2Erk2Erk2
p-Erk1/2
Fig. 9. Phospho MAP kinase activity in SHI-
P2 + ⁄ + and – ⁄ – MEF cells. + ⁄ +and–⁄ – MEF
cells were stimulated as in Fig. 8. Protein
(100 lg) was immunoblotted and probed
with p-Erk1 ⁄ 2 for MAP kinase activity. Total
MAP kinase (Erk2) is shown below.
Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts D. Blero et al.
2518 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS
levels but that it may control the duration and mag-
nitude of stimulated increases in this lipid [25,34].
We did not detect any upregulation of
PtdIns(3,4,5)P
3
by stimulation of the cells with HGF,
b-FGF, IGF-1 or EGF. We excluded any difference in
regulation of PI 3-kinase activity between serum and
IGF-1 stimulated cells as no differences in PI 3-kinase
activity could be detected between SHIP2 + ⁄ + and
– ⁄ – MEF cells (this effect was reversed in the presence
of LY-294002). The reason for not observing an upreg-
ulation of PtdIns(3,4,5)P
3
with every agonist is not
yet understood, however, maximal production of

will only be able to control the PtdIns(3,4,5)P
3
levels
once PTEN is inactivated by oxidation. This mechan-
ism may be dependent on both the type of agonist and
the cell type. In another study, others have recently
reported enhanced PKB activation in response to
M-CSF in fetal liver-derived macrophages prepared
from SHIP2 knockout mice [8].
Our data also suggest that one or several compo-
nents of the serum allows SHIP2 to be effectively
recruited near the sites of PtdIns(3,4,5)P
3
production.
The localization of SHIP2 at the membrane is import-
ant for its lipid phosphatase activity as shown in 3T3-
L1 adipocytes where insulin provokes a redistribution
of SHIP2 from the cytosol to the plasma membrane
fraction following a mechanism which is in part
dependent on PI 3-kinase activity [14]. Moreover, as
discussed above, PTEN is also competing for SHIP2 in
the regulation of PtdIns(3,4,5)P
3
and PtdIns(3,4)P
2
lev-
els and this may affect the kinetics of the phosphoino-
sitides in stimulated cells. The influence of PTEN
activity in this complex pathway is not known but we
clearly established in our study that PTEN expression

[5]. PTEN antibody was from A.G. Scientific, Inc. (San
Diego, CA, USA) Anti-PI 3-Kinase p85 and antiphosphotyr-
osine were from Upstate (AH Veemendaal, the Netherlands).
Anti-Insulin receptor (InsR) and anti IGF-1 antibodies
were kindly provided by K. Siddle (Department of Clinical
Biochemistry, Cambridge University, UK). HGF, IGF-1 and
PDGF were provided by Upstate. LY-294002, PI and phos-
phatidylserine were from Sigma (Bonnem, Belgium). Easi-
tides [c-
32
P] ATP (3000 CiÆmmol
)1
) was from NEM.
[
32
P]Orthophosphate (10 mCiÆmL
)1
) was from Amersham
(Rosendaal, the Netherlands).
PtdIns(3,4,5)P
3
measurements
Cells (1.5 · 10
6
) were cultured in 10% serum overnight.
Cells were washed twice in medium without serum and
twice in medium without either phosphate or serum. They
were labelled for at least 4 h in medium with [
32
P]ortho-

After visceral organ removal, the rest of the body was minced
finely by repetitive syringe aspiration, then washed twice with
1 · NaCl ⁄ Pi and incubated in 500 lL trypsin⁄ EDTA (2.5%
trypsin, 1 mm EDTA) at 37 °C for 60 min. The embryo
fragments were resuspended by adding 1.5 mL of complete
medium (DMEM, 2% streptomycin ⁄ ampicillin, 50 lm
b-mercaptoethanol) with a 2-mL glass pipette. The cells were
dissociated with a 10-mL pipette by adding another 7.5 mL
complete medium. The supernatant was transferred to a T75
culture flask after 2 min resting. MEF were obtained after
incubation of the cells at 37 °C for 2–3 days.
Genotyping of MEF cells
Genotyping of SHIP2 MEF was performed by PCR using
specific primers to amplify the neo gene and a specific exon
deleted in the recombinant allele [18]. The same forward
primer was used for each of the + ⁄ + and – ⁄ – alleles:
5¢-GGGTCTTTGGAGCTGTGGACT-3¢. While specific
reverse primers were used for the + ⁄ + allele:
5¢-CCCAAGTGTCTCCCATCATCC-3¢ and for the – ⁄ –
allele: 5¢-TAAGGGTTCCGGATCTGCC-3¢. The PCR
reaction was performed under the following conditions:
denaturation at 95 °C for 3 min, followed by 40 cycles at
95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and elonga-
tion of 72 °C for 7 min.
Cell lysates, PKB and MAP kinase assay
MEF cells were lysed in 50 mm Tris ⁄ HCl pH 7.4, 1% NP-
40, 0.5% cholate, 0.1% Triton X-100, 1 mm EDTA, 1 mm
EGTA, 50 mm NaF, 20 mm b glycerophosphate, 15 mm
sodium pyrophosphate, 2 mm orthovanadate, 10 nm oka-
daic acid, protease inhibitors (Roche, Vilvoorde, Belgium),

condition) and 50 lL sonicated vesicles of PI and phos-
phatidylserine. When the effect of the PI 3-kinase inhibitor
LY-294002 was tested, it was added to the kinase buffer at
25 lm. The lipids were extracted following a Bligh and
Dyer modified procedure and resuspended in 30 lLof
CHCl
3
⁄ CH
3
OH (1 : 1). Separation of the reaction product
was performed by TLC on a silica plate in acetone ⁄
CH
3
OH ⁄ acetic acid ⁄ H
2
O ⁄ CHCl
3
(30 : 26 : 24 : 14 : 80,
v ⁄ v ⁄ v ⁄ v ⁄ v). The corresponding spots were analysed by
autoradiography.
Acknowledgements
We would like to thank Mrs Colette Moreau, Dr Len
Stephens, Louis Hue, Mark Rider, Franc¸ ois Willer-
main, Fabrice Vandeput, Vale
´
rie Dewaste and Natha-
lie Paternotte for many helpful discussions. This work
was supported by grants of the Fonds de la Recherche
Scientifique Me
´

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