This was also observed in rat vascular smooth muscle cells, where PtdIns3,4,5P3 levels were decreased in SHIP2 transfected cells stimulated by PDGF or IGF-1 [14].. SHIP2 modulated PtdIns
Trang 1in SHIP2-deficient mouse embryonic fibroblasts
Daniel Blero1, Jing Zhang1, Xavier Pesesse1, Bernard Payrastre2, Jacques E Dumont1,
Ste´phane Schurmans3and Christophe Erneux1
1 Interdisciplinary Research Institute (IRIBHM), Universite´ Libre de Bruxelles, Belgium
2 INSERM U563, Departement d’Oncogenese et Signalization dans les Cellules Hematopoietiques, Hoˆpital 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],
particularly in cells that do not express SHIP1 (e.g in
heart, muscle or adipocytes) Tyrosine phosphorylation
of SHIP2 occurs in response to treatment of cells with
various stimuli, e.g epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin or macrophage colony-stimulating factor (M-CSF) but the biological significance of this phosphorylation is unknown [6–8] In 3T3-L1 preadipocytes, SHIP2 translocation to the plasma membrane occurs in response to insulin or PDGF In this model, SHIP2 translocation does not seem to require its tyrosine
Keywords
inositol 5-phosphatase; mouse embryonic
fibroblasts; phosphatidylinositol
3,4,5-trisphosphate; SH2 domain; signal
transduction.
Correspondence
C Erneux, Institute of Interdisciplinary
Research (IRIBHM), Campus Erasme
Building C, 808 Route de Lennik, 1070
Brussels, Belgium
Fax: +32 2 555 4655
Tel: +32 2 555 4162
E-mail: cerneux@ulb.ac.be
Note
D Blero and J Zhang contributed equally to
this work.
(Received 27 July 2004, revised 6 February
2005, accepted 21 March 2005)
doi:10.1111/j.1742-4658.2005.04672.x
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)P3]
in vitro The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P3 levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (–⁄ –) MEF cells derived from knockout mice PtdIns(3,4,5)P3 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)P3 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)P3 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, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P3levels in intact cells
Abbreviations
CHO-IR, chinese hamster ovary cells overexpressing the insulin receptor; EGF, epidermal growth factor; FBS, foetal bovine serum;
FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; MAP, mitogen activated protein; M-CSF, macrophage colony-stimulating factor; MEF, mouse embryonic fibroblast; PDGF, platelet-derived growth factor; PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PtdIns(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P 3 , phosphatidylinositol
3,4,5-trisphosphate; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PTEN, phosphate and tension homolog deleted on chromosome 10; SHIP, SH2 domain containing inositol phosphatase.
Trang 2phosphorylation [9] As phosphatidylinositol
3,4,5-tris-phosphate [PtdIns(3,4,5)P3] is a major intracellular
sig-nal generated by insulin and insulin-like growth factor
(IGF)-1, it has been suggested that SHIP2 is a
physio-logically negative regulator of their signalling Indeed,
overexpression of SHIP2 inhibited insulin-induced
glu-cose uptake and glycogen synthesis in 3T3-L1
adipo-cytes and L6 myotubes [10,11] SHIP2 also appears to
inhibit the insulin-induced phosphorylation of Akt2,
but not Akt1, in 3T3-L1 adipocytes Upon insulin
sti-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
G1 [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]
Removal of exons 1–18 of SHIP2 resulted in a
differ-ent phenotype: the mice were viable and had no
increased insulin sensitivity but they were smaller in
body weight and length, and were highly resistant to
weight gain when placed on a high-fat diet [19] The
reason for this discrepancy between the two
pheno-types is currently not understood but several
explana-tions have been proposed [19]
We and others previously reported that SHIP2
displays inositol 5-phosphatase activity when
PtdIns(3,4,5)P3 and phosphatidylinositol
4,5-bisphos-phate [PtdIns(4,5)P2] were used as substrate in vitro
Inositol tetrakisphosphate was also a substrate of the
enzyme expressed in bacteria [15,20,21] Moreover, both
in COS-7 cells and in chinese hamster ovary cells
over-expressing the insulin receptor (CHO-IR) cells transfected with SHIP2, the levels of PtdIns(3,4,5)P3were decreased
in both EGF and insulin stimulated cells [22,23] This was also observed in rat vascular smooth muscle cells, where PtdIns(3,4,5)P3 levels were decreased in SHIP2 transfected cells stimulated by PDGF or IGF-1 [14] Both PKB and mitogen activated protein (MAP) kinase 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)P3 regulation in SHIP2 –⁄ – cells derived from MEF cells Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P3levels, 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)P2 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
of phosphoinositides (see below) Western blot analy-sis of SHIP2 was performed to confirm the absence
of expression of SHIP2 in –⁄ – MEF cells (Figs 1A and B) The expression of SHIP2 in +⁄ – MEF cells was decreased as compared to wild type (+⁄ +) MEF cells (Fig 1A) as reported previously [18] Although we detected the presence of the IGF-1 receptor in MEF cells by western blotting, the b sub-unit of the insulin receptor was not be seen by this method suggesting that it is either not expressed or was below the detection level of the antibodies used
in our immunodetection method (Fig 1B) The expression of the PtdIns(3,4,5)P33-phosphatase, phos-phatase and tension homolog deleted on chromosome
10 (PTEN) [24,25] was not significantly modified between SHIP2+⁄ + and – ⁄ – MEF cells (Fig 1B) No changes in expression of the regulatory subunits of PI 3-kinase p85 were seen between the two types of cells (data not shown)
Trang 3No change in PtdIns(4,5)P2levels in SHIP2
+/+ and –/– MEF cells
As in vitro, PtdIns(4,5)P2 is also a substrate of SHIP2
[15], we compared the levels of [3H]PtdIns(4,5)P2 and
[3H] phosphatidylinositol 4-phosphate (PtdIns4P) after
labelling the cells with [3H]inositol in the presence of 10% FBS for 72 h: the amount of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P did not change significantly between +⁄ + and – ⁄ – MEF cells (Fig 2A) Similar results were obtained when we labelled the cells with [32P]orthophosphate for more than 4 h In our assay
SHIP2
M E
F SH
IP2
+/ + 1
M E
F SHIP2
+/ + 2
M E
F SH IP2
+/
-M E
F SH IP2
-/ - 2
M E
F SH
IP2
-/ - 1
CHO -IR
150
100
75
250
kDa
SHIP2
InsR
IGF-IR
PTEN
M E
F SH IP2
+ /+
2
M E
F SH IP2
-/ - 1
ME
F SHIP2
+/ +
1
M E
F SH IP2
-/
-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 levels Labelling with [ 3 H] inositol
0 0,2 0,4 0,6 0,8 1 1,2
TIME (minute)
SHIP2+/+
SHIP2-/-0 1 2 3 4 5 6
PtdInsP PtdIns(4,5)P2
SHIP2+/+
SHIP2-/-PtdIns(3,4,5)P 3 levels
0 0,1 0,3 0,4 0,5 0,7 0,9
SHIP2+/+
SHIP2-/-PtdIns(3,4,5)P 3 levels
0 0,2 0,4 0,6 0,8 1 1,2 1,4
SHIP2+/+
SHIP2+/-
SHIP2-/-Fig 2 PtdIns(3,4,5)P3levels in serum stimulated SHIP2 + ⁄ +, + ⁄ – and – ⁄ – MEFs (A) MEF cells were labelled with 50 lCi [ 3 H]inositol for
72 h in the presence of FBS [ 3 H]phosphoinositides were isolated as described The data were normalized with respect to the total radioac-tivity present in the phosphatidylinositol fraction The data are means of triplicates ± SD (B) + ⁄ + and – ⁄ – MEF cells were labelled with [ 32 P]orthophosphate for 4 h and stimulated with 10% serum for various periods of time [ 32 P]PtdIns(3,4,5)P3was isolated as described The data are expressed as a percentage of total [ 32 P]PtdIns(4,5)P2 measured in the same HPLC profile and are means of triplicates ± SD (C) MEF cells were labelled with [32P] for 4 h and stimulated by 10% FBS for 10 min The data are expressed as a percentage of total [32P] PtdIns(4,5)P2measured in the same HPLC profile The data are means of three independent experiments using the two series of MEF cells (1 and 2) ± SD (D) + ⁄ +,+ ⁄ – and – ⁄ – MEF cells were labelled with [ 32 P] and stimulated for 10 min by 10% FBS The data are means of duplicates ± SD The data are representative of two different experiments.
Trang 4of [32P]-labelled lipids, we scraped off together the
region of the TLC containing both [32P]PtdIns(3,4,5)P3
and [32P]PtdIns(4,5)P2, the levels of [32P]-labelled
3-phosphoinositides were normalized with respect to
[32P]PtdIns(4,5)P2
SHIP2 modulated PtdIns(3,4,5)P3levels after
short-term serum stimulation
The levels of PtdIns(3,4,5)P3 were compared between
the two types of cells that had been stimulated by
serum PtdIns(3,4,5)P3 was increased following
stimu-lation of both types of cells by 10% FBS In
res-ponse to the addition of serum, the production of
PtdIns(3,4,5)P3 was upregulated in SHIP2 –⁄ – cells as
compared to +⁄ + cells A maximal effect was seen
between 5 and 10 min after stimulation by 10% serum
(Figs 2B and 3A) This effect was observed in the two
independently prepared MEF cells (Fig 2C) In these experiments, the levels of phosphatidylinositol 3,4-bis-phosphate; [PtdIns(3,4)P2] were not significantly differ-ent between the two types of cells (see below) This probably reflects the complex pathway of PtdIns(3,4)P2 production⁄ degradation in response to FBS via PI 3-kinase, PTEN, SHIP2 and several other phosphati-dylinositol 5-phosphatases [25] The difference in PtdIns(3,4,5)P3 levels between serum stimulated +⁄ + and –⁄ – cells was also observed in heterozygous MEF cells but the effect was intermediate as compared to the +⁄ + and – ⁄ – MEF cells Basal levels of PtdIns(3,4,5)P3 were not different between +⁄ + and –⁄ – cells (Fig 2D)
In contrast to serum, IGF-1 stimulation resulted in maximal production of PtdIns(3,4,5)P3after 2 min and
no significant differences in PtdIns(3,4,5)P3 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)P3 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)P3levels after long-term serum stimulation
Previous studies in MEF cells deficient in PTEN have shown that PtdIns(3,4,5)P3 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)P3 is a physiological substrate of PTEN [26] We therefore measured PtdIns(3,4,5)P3 levels in SHIP2 deficient cells after long-term stimulation by FBS (under the same conditions used in the study of PTEN deficient MEF cells) PtdIns(3,4,5)P3 levels in SHIP2 –⁄ – cells were not significantly different as com-pared to +⁄ + cells after 30 min or 4 h of stimulation
by 5% FBS (Figs 2B and 4, respectively) Therefore,
no significant differences in PtdIns(3,4,5)P3 levels between SHIP2 +⁄ + and – ⁄ – were observed under the conditions where PtdIns(3,4,5)P3 levels were upreg-ulated in PTEN deficient cells
PI 3-kinase activity in SHIP2 +/+ and –/– MEF cells
PI 3-kinase activity in SHIP2 +⁄ + and – ⁄ – cells was determined in both the presence and absence of the PI 3-kinase inhibitor LY-294002 Basal activity was stimula-ted in the presence of 10% FBS or IGF-1 at 10 nm This activity was reversed when lysates were prepared in the presence of LY-294002 (Fig 5) No differences in the
0
0,2
0,4
0,6
0,8
1
1,2
TIME (minute)
TIME (minute)
SHIP2+/+
SHIP2-/-A
+serum
0
0,1
0,2
0,3
0,4
0,5
0,6
SHIP2+/+
SHIP2-/-B
+ IGF-1
Fig 3 PtdIns(3,4,5)P 3 levels in serum and IGF-1 stimulated MEF
cells Time course of [32P] PtdIns(3,4,5)P 3 production in (A) serum
and (B) IGF-1 stimulated MEF cells + ⁄ + and – ⁄ – MEF cells were
labelled with [ 32 P]orthophosphate for 4 h and stimulated with 10%
serum or IGF-1 at 10 n M for the indicated times [32P] PtdIns(3,4,5)P 3
was quantified as before.
Trang 5level of activation was determined in the SHIP2 +⁄ +
and –⁄ – MEF cells at the time of maximal production of
PtdIns(3,4,5)P3, 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)P3
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 inhibiinactiva-tion [10,15,22,23]
Activa-ted PKB was detecActiva-ted 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)P3 and PtdIns(3,4)P2production EGF sti-mulated both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels but the differences in the two lipid levels were not signifi-cant between the two types of cells (SHIP2 deficient or not) IGF-1 at 10 nm stimulated PtdIns(3,4,5)P3 produc-tion but PtdIns(3,4,5)P3 levels were not significantly upregulated in the –⁄ – cells compared to the + ⁄ + cells
-/-Origin
PI3P
Control Control with LY No lysates
A
-/-Origin
PI3P
B
-/-Origin
PI3P
SHIP2 MEF
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.
0
0.05
0.1
0.15
0.2
0.25
+ serum
Fig 4 PtdIns(3,4,5)P3 levels in serum stimulated SHIP2 + ⁄ + and
– ⁄ – MEF cells after long-term stimulation + ⁄ + and – ⁄ – MEF cells
were labelled with [32P] and stimulated with 5% serum for 4 h.
[ 32 P]PtdIns(3,4,5)P3was quantified as before.
Trang 6as shown before in the kinetics of the lipid production
(Fig 3B) In PDGF stimulated cells, PtdIns(3,4,5)P3
levels increased but no significant differences were seen
between +⁄ + and – ⁄ – cells although PtdIns(3,4)P2was
lower in SHIP2 deficient cells as compared to control
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)P2, PtdIns(3,4,5)P3 and ino-sitol tetrakisphoshate in vitro [7,15,21]
SHIP2
A
B
total PKB
p-Erk1/2 Erk2
1 2 3 4 5 6 7 8
Western blotting
PKB assay
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
SHIP2+/+
SHIP2-/-Fig 6 PKB and MAP kinase activities in SHIP2 + ⁄ + and – ⁄ – MEF
cells (A) SHIP2 + ⁄ + (lane 1,2 and 5,6) and – ⁄ – (lane 3,4 and 7,8)
MEF cells were stimulated by 10% FBS for 10 min (lane 1–4
unstimulated, 5–8 stimulated by FBS) Protein (20 lg) wase
immuno-blotted with the indicated antibodies Phosphorylation was assayed
by using phospho-specific antibodies against p-Thr308 or p-Ser473
for PKB and p-Erk1 ⁄ 2 for MAK kinase activities Western blot
against total PKB or Erk2 is shown below Results are
representa-tive of three experiments (B) PKB activities determined after
immunoprecipitation of endogenous PKB as described Protein cell
lysate (100 lg) was used in the assay Results are means of
dupli-cates ± SD.
SHIP2 MEF +/+ - / - +/+ - / - +/+ - / - +/+ /
2
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 abspres-ence of LY-294002 (25 l M ) Protein (20 lg) was immunoblotted Phosphorylation of PKB was assayed in the pres-ence of antibodies against p-Ser473 and total PKB is shown below.
PtdIns(3,4)P 2 levels in MEF cells (5 min stimulation)
0 0,5 1 1,5 2 2,5 3 3,5
50ng/ml HGF 15ng/ml β-FGF 100ng/ml IGF-1 1nM
30ng/ml
SHIP2+/+
SHIP2-/-0 0,2 0,4 0,6 0,8 1 1,2
S B
% 0 G E
50 m / G H
1n m /
β-FG
00n g/
l IG -F
nM G
I F-1 M
P F G 3 g/
l m
SHIP2+/+
SHIP2-/-PtdIns(3,4,5)P 3 levels ( 5 min stimulation)
Fig 8 PtdIns(3,4,5)P3and PtdIns(3,4)P2levels 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
1 and 10 n M or PDGF 30 ngÆmL)1 for 5 min [ 32 P]PtdIns(3,4,5)P3 (upper panel) and [32P]PtdIns(3,4)P 2 (lower panel) are expressed as
a percentage of total [ 32 P]PtdIns(4,5)P2 and are means of dupli-cates ± SD NS ¼ non stimulated cells.
Trang 7The ability of SHIP2 to be a physiological regulator
of PtdIns(3,4,5)P3 is often assumed based on a similar
function attributed to SHIP1 [27,28] Two SHIP2
knockout mice have now been reported The first
knockout mice showed an increased sensitivity to
insu-lin although it was later recognized that another gene
Phox2a was also deleted in the targeting construct
together with the 19–29 exons of SHIP2 [18,29] It is
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)P3 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
SHIP2 is unclear in preadipocytes [9] and multiple
phosphorylation sites have been identified, e.g in
Jurkat cells [33] Thus, the biochemistry of SHIP2 in
the insulin (and probably other signalling cascades) is
not fully understood
We were interested to measure PtdIns(3,4,5)P3 lev-els in SHIP2 depleted cells As SHIP2 did not appear
to be specific for a given signalling pathway in cellu-lar models, we compared the effects of a series of agonists including EGF, PDGF and IGF-1 that had been previously used in SHIP2 overexpression studies We also compared our data with PTEN which is often presented as the principal regulator of PtdIns(3,4,5)P3 levels [25] This is the first report showing a direct comparison of PtdIns(3,4,5)P3 pro-duction in SHIP2 +⁄ + and – ⁄ – cells PtdIns(3,4,5)P3 levels [but not PtdIns(4,5)P2 levels] were potentiated
in serum stimulated SHIP2 –⁄ – MEF cells as com-pared to +⁄ + cells; this effect was not observed in unstimulated cells, or after long-term stimulation (i.e
1 or 4 h) of the cells PKB activity (but not MAP 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)P3 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)P3 levels and PKB activity measured in our study is a consequence of an effect on PtdIns(3,4,5)P3 dephosphorylation Stambolic et al [26] reported that PtdIns(3,4,5)P3 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)P3 levels were seen in SHIP2 depleted cells as compared to +⁄ + cells either at the basal level or after long-term stimulation We found that SHIP2 acted after short-term (5–10 min) serum stimulation as a modulator of PtdIns(3,4,5)P3 levels Previous data obtained with SHIP1 also indicated that SHIP1 does not regulate basal PtdIns(3,4,5)P3
IGF -1 1 M
IGF 1 10nM PDGF 30ng/ml FBS 10%
control
IGF -1
1 nM IGF 1 10nM PDGF 30ng/ml FBS 10%
control Erk2
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.
Trang 8levels 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)P3 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)P3 with every agonist is not
yet understood, however, maximal production of
PtdIns(3,4,5)P3 was observed after 5 min stimulation
by serum and after only 2–3 min by IGF-1; the
ampli-tude of the PtdIns(3,4,5)P3 production was also not
comparable being higher for serum as compared to
IGF-1 (as shown in Fig 3) Finally, we cannot ignore
the fact that our method does not allow the
measure-ment of minor or local changes in PtdIns(3,4,5)P3
levels that are observed only in the presence of serum
These observations could contribute to the specificity
we have observed with serum However, in addition to
serum, we found that short-term H2O2 treatment of
the SHIP2 –⁄ – MEF cells also upregulates the
phos-phorylation of PKB (J Zhang, unpublished data) This
observation suggests the following model: it is possible
that serum is producing some reactive oxygen species
which could be responsible for the inactivation of
PTEN The production of reactive oxygen species in
response to growth factors or insulin and inactivation
of PTEN has been recently reported by others [35,36]
In this model, we assume that SHIP2 is less active than
PTEN in terms of enzymatic activity and that SHIP2
will only be able to control the PtdIns(3,4,5)P3 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)P3 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)P3 and PtdIns(3,4)P2
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
is unchanged between SHIP2 wild type and SHIP2 deficient cells
In contrast to data obtained in cells transfected with SHIP2, no difference in MAP kinase activity was observed between serum stimulated SHIP2 +⁄ + and –⁄ – cells; this suggests that the SHIP2 pathway does not interfere with MAP kinase activity in serum stimulated cells
In conclusion, our data are consistent with SHIP2 affecting the transient control of PtdIns(3,4,5)P3 levels which influences PKB activity, at least in cells stimula-ted by serum The specificity of PtdIns(3,4,5)P3 hydro-lysis by SHIP2 with regard to serum stimulation and the short-term kinetics of the SHIP2 action add an interesting twist to the sophistication of these signal-ling systems This concept, is quite reminiscent of some
of the characteristics of Ca2+signalling, e.g the differ-ent aspects of neuronal differdiffer-entiation encoded by the frequency of Ca2+transients [37]
Experimental procedures
Materials
SHIP1 and SHIP2 antibodies have been described previously [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-32P] ATP (3000 CiÆmmol)1) was from NEM [32P]Orthophosphate (10 mCiÆmL)1) was from Amersham (Rosendaal, the Netherlands)
PtdIns(3,4,5)P3measurements
Cells (1.5· 106
) 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-phosphate (250 lCiÆmL)1) but without serum Cells were stimulated with various agents as indicated in the text The reaction was terminated by 5 mL cold NaCl⁄ Pi Cells were lysed in 3.75 mL 2.4 N HCl Lipids were extracted
in 3 mL methanol and 4.5 mL CHCl3 After TLC and deacylation of the phosphoinositides, separation was per-formed by HPLC on Whatman SAX columns (Leuven, Belgium) [23] Radioactivity was estimated with an online detector from Raytest (Straubenhardt, Germany)
Trang 9PtdIns(4,5)P2 was also determined by labelling the cells
with [3H] inositol as reported [38] The various
3-phos-phorylated phosphoinositides standards were prepared in
insulin stimulated CHO-IR cells or in platelets as reported
previously [22,39]
Preparation of MEF cells
Primary MEF cells were isolated from 14-day postcoitus
C57BL6 mouse embryos [18] Embryos were surgically
removed and separated from maternal tissues following
pro-tocol approved by the Ethics Committee for animals The
head of each embryo was removed and kept for genotyping
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 37C 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 37C 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 95C for 3 min, followed by 40 cycles at
95C for 30 s, 60 C for 30 s, 72 C for 30 s and
elonga-tion of 72C 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),
0.1 m NaCl Activated PKB was detected using
phospho-specific antibodies against T308 or S473 Activated MAP
kinase was detected using phospho-p44⁄ 42 MAP (Erk1 ⁄ 2)
kinase antibody (Cell Signalling, Leusden, the Netherlands)
Antibodies against total PKB and MAP kinase (Erk2) were
from Cell Signalling For PKB assay, immunoprecipitation
was performed with anti Akt1⁄ PKB (Upstate) following the
protocol provided PKB activities were determined in the presence of 30 lm Crosstide substrate peptide and [c-32P]ATP Phosphorylated substrate was measured on P81 phosphocellulose papers
Measurement of PI 3-kinase activity
Cell lysates of serum or IGF-1 stimulated MEF cells (2· 106 cells per condition) were immunoprecipitated with
2 lL antiphosphotyrosine antibodies overnight at 4C The immunoprecipitates were washed twice in 50 mm Tris⁄ HCl
pH 7.4, 200 mm NaCl, 0.1% Brij, protease inhibitors (Roche) and once in the kinase reaction buffer (see below) The pellet ( 30 lL) was incubated at 37 C for 30 min in the presence of 80 lL 2· kinase buffer containing 100 mm Tris⁄ HCl pH 7.4, 200 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 200 lm ATP together with [c-32P]ATP (10 lCi per 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 lL of CHCl3⁄ CH3OH (1 : 1) Separation of the reaction product was performed by TLC on a silica plate in acetone⁄
CH3OH⁄ acetic acid⁄ H2O⁄ CHCl3 (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´dicale, Action de Recherche Concerte´e
of the Communaute´ Franc¸aise de Belgique and INSERM-Communaute´ Franc¸aise de Belgique exchange contract This work was executed in the framework of research network IAPV-O5 (Belgium Science Policy) Daniel Blero and Xavier Pesesse are Charge´ de Recherche FNRS
References
1 Damen JE, Liu L, Rosten P, Humphries RK, Jefferson
AB, Majerus PW & Krystal G (1996) The 145-kDa pro-tein induced to associate with Shc by multiple cytokines
is an inositol tetraphosphate and phosphatidylinositol 3,4,5- triphosphate 5-phosphatase Proc Natl Acad Sci USA 93, 1689–1693
2 Ishihara H, Sasaoka T, Hori H, Wada T, Hirai H, Har-uta T, Langlois WJ & Kobayashi M (1999) Molecular
Trang 10cloning of rat SH2-containing inositol phosphatase 2
(SHIP2) and its role in the regulation of insulin
signal-ing Biochem Biophys Res Commun 260, 265–272
3 Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold
A & Rohrschneider LR (1996) p150Ship, a signal
trans-duction molecule with inositol
polyphosphate-5-phos-phatase activity Genes Dev 10, 1084–1095
4 Pesesse X, Deleu S, De Smedt F, Drayer L & Erneux C
(1997) Identification of a second
SH2-domain-contain-ing protein closely related to the phosphatidylinositol
polyphosphate 5-phosphatase SHIP Biochem Biophys
Res Commun 239, 697–700
5 Muraille E, Pesesse X, Kuntz C & Erneux C (1999)
Dis-tribution of the src-homology-2-domain-containing
ino-sitol 5- phosphatase SHIP-2 in both non-haemopoietic
and haemopoietic cells and possible involvement of
SHIP-2 in negative signalling of B-cells Biochem J 342,
697–705
6 Habib T, Hejna JA, Moses RE & Decker SJ (1998)
Growth factors and insulin stimulate tyrosine
phosphory-lation of the 51C⁄ SHIP2 protein J Biol Chem 273,
18605–18609
7 Wisniewski D, Strife A, Swendeman S,
Erdjument-Bro-mage H, Geromanos S, Kavanaugh WM, Tempst P &
Clarkson B (1999) A novel SH2-containing
phosphati-dylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is
constitutively tyrosine phosphorylated and associated
with src homologous and collagen gene (SHC) in
chronic myelogenous leukemia progenitor cells Blood
93, 2707–2720
8 Wang YJ, Keogh RJ, Hunter MG, Mitchell CA, Frey
RS, Javaid K, Malik AB, Schurmans S, Tridandapani S
& Marsh CB (2004) SHIP2 is recruited to the cell
mem-brane upon macrophage colony-stimulating factor
(M-CSF) stimulation and regulates M-CSF-induced
signaling J Immunol 173, 6820–6830
9 Gagnon A, ArtemenkoY, Crapper T & Sorisky A (2003)
Regulation of endogenous SH2 domain-containing
inositol 5-phosphatase (SHIP2) in 3T3-L1 and human
preadipocytes J Cellular Physiol 197, 243–250
10 Sasaoka T, Hori H, Wada T, Ishiki M, Haruta T,
Ishi-hara H & Kobayashi M (2001) SH2-containing inositol
phosphatase 2 negatively regulates insulin- induced
gly-cogen synthesis in L6 myotubes Diabetologia 44, 1258–
1267
11 Wada T, Sasaoka T, Funaki M, Hori H, Murakami S,
Ishiki M, Haruta T, Asano T, Ogawa W, Ishihara H &
Kobayashi M (2001) Overexpression of SH2-containing
inositol phosphatase 2 results in negative regulation of
insulin-induced metabolic actions in 3T3-L1 adipocytes
via its 5¢-phosphatase catalytic activity Mol Cell Biol
21, 1633–1646
12 Sasaoka T, Wada T, Fukui K, Murakami S, Ishihara
H, Suzuki R, Tobe K, Kadowaki T & Kobayashi M
(2004) SH2-containing inositol phosphatase 2
predomi-nantly regulates Akt2, and not Akt1, phosphorylation
at the plasma membrane in response to insulin in 3T3-L1 adipocytes J Biol Chem 279, 14835–14843
13 Hori H, Sasaoka T, Ishihara H, Wada T, Murakami S, Ishiki M & Kobayashi M (2002) Association of SH2-containing inositol phosphatase 2 with the insulin resis-tance of diabetic db⁄ db mice Diabetes 51, 2387–2394
14 Sasaoka T, Kikuchi K, Wada T, Sato A, Hori H, Murakami S, Fukui K, Ishihara H, Aota R, Kimura I
& Kobayashi M (2003) Dual role of Src homology domain 2-containing inositol phosphatase 2 in the regu-lation of platelet-derived growth factor and insulin-like growth factor I signaling in rat vascular smooth muscle cells Endocrinology 144, 4204–4214
15 Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM, Moodie S & Stokoe D (2000) 5¢ phos-pholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells Mol Cell Biol 20, 6860–6871
16 Prasad N, Topping RS & Decker SJ (2001) SH2-con-taining inositol 5¢-phosphatase SHIP2 associates with the p130 (Cas) adapter protein and regulates cellular adhesion and spreading Mol Cell Biol 21, 1416–1428
17 Prasad N & Decker SJ (2005) SH2-containing 5¢ inositol phosphatase SHIP2 regulates cytoskeleton organization and ligand-dependent downregulation of the epidermal growth factor receptor J Biol Chem 280, 13129–13136
18 Clement S, Krause U, Desmedt F, Tanti JF, Behrends
J, Pesesse X, Sasaki T, Penninger J, Doherty M, Mala-isse W, Dumont JE, Marchand-Brustel Y, Erneux C, Hue L & Schurmans S (2001) The lipid phosphatase SHIP2 controls insulin sensitivity Nature 409, 92–97
19 Sleeman MW, Wortley KEV, Lai KM, Gowen LC, Kintner J, Kline WO, Garcia K, Stitt TN, Yancopoulos
GD, Wiegand SJ & Glass DJ (2005) Absence of the lipid phosphatase SHIP2 confers resistance to diary obesity Nat Med 11, 199–205
20 Giuriato S, Blero D, Robaye B, Bruyns C, Payrastre B
& Erneux C (2002) SHIP2 overexpression strongly reduces the proliferation rate of K562 erythroleukemia cell line Biochem Biophys Res Commun 296, 106–110
21 Pesesse X, Moreau C, Drayer AL, Woscholski R, Par-ker P & Erneux C (1998) The SH2 domain containing inositol 5-phosphatase SHIP2 displays phosphatidylino-sitol 3,4,5-trisphosphate and inophosphatidylino-sitol 1,3,4,5- tetraki-sphosphate 5-phosphatase activity FEBS Lett 437, 301–303
22 Blero D, De Smedt F, Pesesse X, Paternotte N, Moreau
C, Payrastre B & Erneux C (2001) The SH2 domain containing inositol 5-phosphatase SHIP2 controls phos-phatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin Biochem Biophys Res Com-mun 282, 839–843
23 Pesesse X, Dewaste V, De Smedt F, Laffargue M, Giur-iato S, Moreau C, Payrastre B & Erneux C (2001) The