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INTRODUCTION |
Tumor necrosis factor
(TNF)1 is involved in many
disease-related processes in the organism, including inflammation,
anti-viral, anti-cancer, and immune responses (1). In combination with inhibitors of transcription or translation, TNF can induce apoptosis in
sensitive cells in cell culture. This apoptosis is initiated by binding
TNF to its plasma membrane receptors, which belong to the death
receptor family. After death receptors have been triggered, the adaptor
molecules, including FADD and TRADD and the receptor proximal caspase
8, are engaged to form a death-inducing signaling complex
(DISC). Upon recruitment to the DISC, caspase 8 becomes
activated and mediates cleavage of procaspase 3, starting a chain of
events that results in apoptotic death (2).
The key role played by TNF pathway in many pathological processes
indicates the importance of its control by pharmacological approaches
(3). Rational design of such approaches requires identification of
cellular factors involved in regulation of TNF-mediated death, some of
which have been already determined. Pro-apoptotic function of TNF can
be counterbalanced by simultaneous activation of anti-apoptotic NF-
B
pathway, which can effectively prevent initiation of a death cascade
through transcriptional induction of a number of apoptosis inhibitors
(4). NF-
B is functionally linked to Akt signaling pathway, an
important sensor of environmental conditions (i.e.
availability of growth factors) strongly contributing to the "to die
or not to die" decision. It can greatly reduce suicidal intentions of
the cell in the presence of growth factors and other ligands specific
to healthy growth conditions and natural microenvironments (5).
The signaling from phosphoinositide 3-kinase (PI3K) to the
protein kinase Akt is an evolutionary conserved ancient pathway that
controls organism life span in invertebrates and cell survival and
proliferation in mammals (6). Many cell-surface receptors induce
production of second messengers, like phosphatidylinositol 3,4,5-trisphosphate (PIP3), that convey signals to the
cytoplasm from the cell surface. PIP3 signals activate
3-phosphoinositide-dependent protein kinase-1, which
in turn activates the kinase Akt, also known as protein kinase B. Akt
promotes cell survival and opposes apoptosis by a variety of routes,
including the activation of NF-
B through phosphorylation of
inhibitory
B kinase (7).
Both NF-
B and Akt signaling are integrated into a network of
cellular regulatory pathways through numerous components, only some of
which have been identified. To determine new regulators of TNF
apoptotic pathway, we used the genetic suppressor elements (GSE)
approach, a functional genetic methodology designed for cloning genes
associated with recessive phenotypes (8, 9). It was successfully used
before for identification of genes involved in negative control of cell
growth, including drug sensitivity and candidate tumor suppressor genes
(8, 10, 11, 12). GSEs act by encoding either inhibitory antisense RNA
or dominant negative truncated proteins. They are isolated from
expression libraries of randomly fragmented cDNAs by functional
selection. GSEs suppressing TNF-induced apoptosis would likely be
derived from and act against the genes that could be important for
TNF-mediated death and therefore can be used for identification and cloning.
Among genetic elements protecting cells from TNF-specific apoptosis, we
isolated a GSE that encoded the cytoplasmic part of the inhibitory
receptor SHPS1 (the mouse homologue of human SIRP
) (13, 14), known
to bind and activate the SH2 domain containing protein tyrosine
phosphatases, SHP-1 and SHP-2, and inositol phosphatase, SHIP (15).
These phosphatases are known to be involved in cytoplasmic signaling
downstream of a variety of cell surface receptors and to be capable of
modulating NF-
B (16). In fact, ectopic expression of
SHPS-1/SIRP
-derived GSE resulted in a strong
Akt-dependent activation of NF-
B that is likely to be
responsible for the GSE-mediated TNF resistance. Overexpression of
full-length SHPS-1/SIRP
protein caused effects opposite to that of
the GSE, indicating that the isolated element acts through a dominant
negative mechanism. These observations suggest that SHPS-1/SIRP
is a
natural negative regulator of NF-
B signaling, presumably involved in
the control of cell response to a variety of stimuli acting through
this important pathway.
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EXPERIMENTAL PROCEDURES |
Cells, Transfection, and Retrovirus Infection--
A4
(p53-deficient mouse embryo fibroblasts transformed by E1a+ras),
NIH3T3, NIH3T3-derived amphotropic and ecotropic packaging cells GP+E86
and GP+envAm12 (17), and HeLa, 293, and Ecopack (Clontech) cell lines were cultured in Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 10%
fetal calf serum. The transfections of NIH3T3, HeLa, 293, and Ecopack
cells were conducted with LipofectAMINE PLUS reagent (Invitrogen)
according to the provider's protocols. Retroviral infection was
accomplished by transferring virus-containing medium
supplemented with 4 µg/ml polybrene (Sigma).
Plasmids and Libraries--
The preparation of the GSE library
from poly(A)+ RNA of NIH3T3 cells was previously described
(8). The library was made in pLNCX retroviral expression vector (18)
and contains 3 × 107 independent clones expressing
random 150-400-bp fragments of normalized cDNA. Synthetic adapters
providing initiator codons upstream of the insert in all reading frames
flank each fragment. GSEs were isolated from selected cells by
RT-PCR and recloned into pCR2.1 vector (Invitrogen) for
sequencing. GSE2-1 and GSESIRP, representing the entire C-terminal
part of SHSP1/SIRP
, were synthesized by RT-PCR from total RNA of
NIH3T3 and HeLa cells, respectively, using 5'-GAATTCTGCAACCATGGAACAG-3'
sense and 5'-GGATCCATCACTTCCTCTGGACCT-3' antisense primers for GSE2-1
and 5'-GAATTCTCGCAACCATGGGACAG-3' sense and
5'-GGATCCATCACTTCCTCGGGACCTG-3' antisense primers for GSESIRP. The
targeted mutagenesis, which converted tyrosines to phenylalanines
codons, was done by PCR. For functional testing, all the generated
fragments were cloned into pLXSN retroviral expression vector, also
conferring G418 resistance. The complete coding region of full-length
SIRP
cDNA was recloned from Expressed Sequence Tag
accession numbers AI357578 and BE514786 into pcDNA3 expression
vector (Invitrogen). cDNA for the S32A/S36A mutant of IkB
(super-repressor mutant form of IkB) in pBabePuro expression vector was
a gift from Dr. S. S. Makarov (19).
GSE Library Screening for TNF Resistance-conferring
Elements--
2 × 106 ECOPACK retrovirus packaging
cells were transfected with 3 µg of GSE library or insert-free pLNCX
plasmid (control) by standard LipofectAMINE PLUS reagent protocol. The
virus-containing medium from transfected cells was transferred on
2 × 106 NIH3T3 amphotropic packaging cells followed
by G418 selection. 5 × 105 G418-resistant NIH3T3
amphotropic cells per plate were treated with TNF (0.01 ng/ml) in the
presence of 1 µg/ml cycloheximide (CHI). Virus-containing medium from
the cells that survived TNF treatment (8-10% of the infected
population) was transferred to 2 × 106 NIH3T3
ecotropic packaging cells that were subjected to the second round of
selection. We alternated amphotropic and ecotropic packaging cells in
this screening to improve the efficiency of infection with retroviruses
(ecotropic packaging cells are resistant to infection with ecotropic
virus, as amphotropic packaging cells are to amphotropic virus). The
last round of selection was done on NIH3T3 cells that are more
sensitive to TNF than packaging cell lines. After 3-4 rounds of
selection, a significant increase in the proportion of TNF-resistant
cells was observed in the population infected by the library-carrying
but not in control insert-free virus. Library inserts from NIH3T3 cells
surviving after TNF/CHI treatment were isolated by RT-PCR, using two
subsequent PCR reactions with two sets of nested primers to increase
specificity of the GSE rescue. The first round of PCR was done with
primers specific to the pLNCX vector sequences flanking the inserts,
5'-CCAAGCTTTGTTTACATCGATG-3' (sense) and 5'-ATGGCGTTAACTTAAGCTGCTT-3'
(antisense), followed with the second PCR that involved the
sense-oriented strand of the adapter 5'-AATCATCGATGGATGGATGG-3'.
NF-
B Luciferase Reporter Assay--
The efficiency of NF-
B
transcriptional activity was estimated using reporter plasmid
containing minimal promoter of the c-fos gene (20)
combined with two oligonucleotides corresponding to NF-
B-binding
sites from human immune deficiency virus long terminal repeat
(21). The efficiency of transfection in luciferase assay experiments was estimated by
-galactosidase expression from a co-transfected pcDNA3-lacZ plasmid, and the results of luciferase assays were normalized accordingly.
Western Immunoblotting--
Total protein extracts from 2 × 106 293 cells expressing full-length SIRP
, GSE2-1,
GSESIRP, and GSE1FSIRP proteins in 50 µl of RIPA buffer (150 mM NaCl, 1% SDS, 10 mM Tris, pH8.0, 1% sodium deoxycholate, 1% Nonidet P-40) with the protease inhibitor mixture (SIGMA) were separated by electrophoresis in 4-20% precast
polyacrylamide gels with SDS (Novex) and transferred to nylon
polyvinylidene difluoride membranes Hybond P (Amersham Biosciences).
Immune complexes were visualized by enhanced chemiluminescence (ECL,
Amersham Biosciences) after incubation with primary rabbit polyclonal
anti-SIRP
/SHPS-1 antibodies (Upstate Biotechnology). For Akt
phosphorylation analysis, 2 × 106 293 cells were
lysed in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 125 µM sodium
ortovanadate (inhibitor of protein phosphatases) (all from Sigma).
Protein concentrations in supernatants were determined with the Bio-Rad
protein assay kit. 40 µg of each protein sample were subjected to
SDS-PAGE electrophoresis 4-20% and transferred to polyvinylidene
difluoride membrane (Amersham Biosciences). The membrane was incubated
with rabbit polyclonal phospho-Akt (Ser473) primary
antibodies (Cell Signaling Technology) or, for quantitation, goat
polyclonal anti-actin antibodies (Santa Cruz). Horseradish peroxidase-conjugated secondary anti-rabbit antibodies were purchased from Cell Signaling Technology, and anti-goat antibodies were purchased
from Santa Cruz. After extensive washing in phosphate-buffered saline,
the membrane was developed with ECL (PerkinElmer Life Sciences).
Quantitation of the data was performed using Quantity One software from
Bio-Rad.
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RESULTS |
Functional Selection of GSEs Protecting from TNF-induced
Apoptosis--
The scheme explaining the screening of the retroviral
GSE library of normalized fragmented cDNA of NIH3T3 is shown in
Fig. 1A and is fully addressed
under "Experimental Procedures." 2 × 106
library-producing NIH3T3-derived packaging cells sensitive to TNF were
treated with TNF in the presence of inhibitor of translation CHI
under conditions that resulted in killing >90% of the cells. After
expansion of the surviving population, virus produced by these cells
(and presumably enriched in clones conferring TNF resistance) was
transferred to another type of packaging cells permissive for the
infection with the isolated virus, and treatment was repeated. Up to
four rounds of selection were applied before virus transfer became
evidently capable of conferring TNF resistance to NIH3T3 cells as
compared with control vector virus subjected to the same selection
procedure (20-25% versus 2-3% of TNF-resistant cells
among infected NIH3T3). At this stage, selected GSE inserts isolated by
RT-PCR were recloned and sequenced. 40% of all rescued GSE clones in
one of the lines of screening contained the same sequence, named GSE2.
The proportion of this sequence, undetectable in the original GSE
population before TNF selection, gradually increased with each next
round of selection, as determined by Southern blot hybridization of PCR
products of the mixture of cDNA GSE inserts isolated by PCR at
different stages of screening (Fig. 1B). This GSE was
subjected to a more detailed analysis.

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Fig. 1.
Isolation of SHPS-1 fragment conferring
resistance to TNF. A, scheme of selection of
GSEs protecting against TNF-induced apoptosis. B, enrichment
of GSE2-expressing cells during selection against TNF-specific
apoptosis. The results of Southern hybridization of RT-PCR products
generated using primers specific for LNCX vector sequences flanking the
library inserts. 1 µg of total RNA from cells infected with GSE
library before TNF selection (0) and after 1, 2, or 3 rounds
of selection (lanes 1-3) was used for RT-PCR. C,
the amino acid sequence of SHPS-1 (in bold) aligned with the
GSEs isolated or generated. The cytoplasmic part of the protein is
underlined. Asterisks mark the homologous amino
acids between mouse and human proteins. Bold letter in
GSESIRP marks the tyrosine substitution with phenylalanine in
GSE1FSIRP.
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Sequence analysis of GSE2 showed that it represents a sense-oriented
fragment of cDNA encoding a cytoplasmic part of a known protein,
inhibitory receptor SHPS-1 (accession number NM007547). (The human
homologue is named signal regulatory protein
or SIRP
). The
SHPS-1/SIRP
corresponding open reading frame in GSE2 starts from the
second initiator codon of the adapter and encodes 99 amino acid
residues from the cytoplasmic part of SHPS-1, with the exception of the
very C-terminal part that includes the last C-terminal tyrosine site
for phosphorylation (Fig. 1C). Structurally, SHPS-1/SIRP
belongs to the immunoglobulin family of receptors. When activated by
Src-mediated phosphorylation of a specific tyrosine residue,
SHPS-1/SIRP
can affect the signaling pathways between epidermal
growth factor receptor and PI3K (22) through binding the SH2-domain
containing phosphotyrosine phosphatases SHP-1 and SHP-2. No connection
has been reported so far between SHPS-1/SIRP
and
TNF-dependent apoptosis.
Cytoplasmic Domain of SHPS-1/SIRP
Protects Mouse
Fibroblasts from TNF-induced Apoptosis--
GSE2 in the selected clone
was in sense orientation and could potentially express the majority of
the cytoplasmic part of SHPS-1/SIRP
if translated from the second
ATG codon of the adapter. However, two other start codons in
alternative frames could, in principle, initiate the translation of
SHPS-1/SIRP
-unrelated peptides, which could be responsible for the
observed effect. To rule out this possibility, we modified the GSE2
sequence to eliminate two alternative initiator codons and cloned it
into another retroviral vector (pLXSN) under the control of the long terminal repeat promoter. In parallel, we synthesized by RT-PCR and
cloned into the same vector the fragment of SHPS-1/SIRP
cDNA that encodes the entire C-terminal region of the protein, supplying it
with the initiator ATG codon (GSE2-1) (Fig. 1C).
All these constructs, including insert-free vector, were converted into
retroviruses by transfection of packaging cells and transduced into two
mouse cell lines (NIH3T3 and A4) that were then tested for TNF
resistance. Only GSE2 and GSE2-1 efficiently protected the infected
cells from TNF-specific apoptosis (Fig. 2). Cells infected with GSE2-1 in
antisense orientation showed the same sensitivity to TNF as the cells
infected with empty pLXSN vector and the original cells. No difference
in the potency of TNF protection was found between GSE2 and GSE2-1,
indicating that the last tyrosine of SHPS-1/SIRP
, which was reported
to be important for SHPS-1/SIRP
function (23), is dispensable in the
GSE activity. Thus, biological activity of the isolated GSE is
indeed the function of the C-terminal fragment of SHPS-1/SIRP
protein expression.

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Fig. 2.
Ectopic expression of GSE2 protects cells
from TNF-induced apoptosis. A, NIH3T3 cells were
infected with "empty" LXSN retrovirus and with retrovirus
expressing GSE2 and selected for G418 resistance. The resulting
populations were treated for 8 h with TNF in the presence of CHI
(TNF/CHI) and stained with DAPI
(4,6-diamidino-2-phenylindole), which reveals apoptotic cells by
bright spots of condensed chromatin. B, relative survival of
NIH3T3 cells after 8 h of TNF/CHI treatment. The cells remaining
on the plates after treatment were fixed with formaldehyde and stained
with methylene blue. The dye was extracted with 0.1 M HCl
and measured at 560 nm. The average data of two experiments are
presented. u/t, untreated cells. C,
GSE2-1, a modified version of GSE2, also protects mouse cells from
TNF. Mouse fibroblast cells, A4, were infected with retrovirus
expressing GSE2-1 in sense (1, 2) and antisense orientation (3, 4).
The transduced population was either untreated
(u/t) (panels 1 and 3) or
treated with TNF/CHI for 10 h (panels 2 and
4), followed by a 24-hour incubation in
TNF/CHI-free medium. D, quantitation of the results of the
experiment shown in panel C using methylene blue assay. Only
expression of sense-oriented GSE2-1 protected cells against
TNF-induced apoptosis. TNF sensitivity of GSE2-1AS (antisense
orientation)-expressing cells was at the level of the population
transduced with insert-free vector.
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Cytoplasmic Domain of SHPS-1/SIRP
Can Activate
NF-
B--
TNF is known to stimulate activation of NF-
B
possessing pro-inflammatory and anti-apoptotic functions (24, 25).
NF-
B activation by TNF and platelet-derived growth factor requires Akt serine-threonine kinase (26, 27). SIRP
was reported to have
negative regulatory effects on cellular responses to growth factors
(13) and to inhibit EGF-induced PI3K activation (22). Because SIRP
affects the PI3K/Akt pathway, we speculated that it can modulate TNF
signaling through Akt-dependent NF-
B activation. To test
this hypothesis we analyzed NF-
B activity in the cells overexpressing GSE2-1 and full-length SIRP
, using specific reporter constructs expressing NF-
B-dependent luciferase. The
results of these experiments are presented in Fig.
3A. They show that GSE2-1
stimulates basal activity of NF-
B up to 8 times. At the same time,
overexpression of the full-length SIRP
protein had the effect
opposite to that of the GSE, causing a decrease in the activity of
NF-
B (Fig. 3A). Neither construct had any effect on the
expression of luciferase reporter with minimal promoter (data not
shown).

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Fig. 3.
Ectopic expression of GSE2-1 and full-length
SIRP has opposite effects on transcriptional activity of
NF- B. A, HeLa, NIH3T3, and 293 cells were
cotransfected with the combination of 50 ng of NF- B-responsive
luciferase reporter construct and 1 µg of empty vector (control), or
1 µg of GSE2-1-expressing plasmid, or 1 µg of full-length SIRP
expression plasmid, and 50 ng of -galactosidase-expressing plasmid
as a control for efficiency of transfection. Data represent luciferase
activity relative to the level of control cells after normalization to
the efficiency of transfection. The average data of four experiments
are presented. B, the super-repressor mutated form of I B
(SRI B) abolishes TNF- and GSE2-1-dependent activation
of NF- B. 293 cells were transfected as described in the legend to
panel A. Cells were lysed 4 h after treatment with 0.01 ng/ml TNF followed by luciferase detection in the lysate. 500 ng of
SRI B expression vector were used for transfection. Bars
represent average results of two experiments. C, detection
of truncated and full-length SIRP proteins by Western
immunoblotting. 293 cells were cotransfected with 1 µg of plasmids
expressing corresponding GSE or full-length SIRP and 50 ng of green
fluorescence protein-expressing plasmid (control of transfection
efficiency). Total protein extracts were made in 50 µl of RIPA
solution. 10 µl of protein extract was used for Western blotting
analysis. Lane 1, GSESIRP; lane 2, GSE2-1;
lane 3, GSE1FSIRP; lane 4, SIRP ; lane
5, untransfected cells extract. Notice that the expression level
of exogenous full-length SIRP always exceeded that of the endogenous
gene, whereas the expression of the truncated protein was close to the
endogenous gene. The mobility of GSE2-1 differed from GSESIRP and
GSE1FSIRP because GSE2-1 is the fragment of the mouse homologue of
human SIRP .
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The mutated form of I
B known as "super-repressor," which can
effectively inhibit NF-
B transcriptional activity at the step of
translocation of NF-
B to the nucleus (19), has often been used to
prove the NF-
B-specificity of the studied effects (19, 27). We
expressed the super-repressor form of I
B in combination with GSE2-1
and found that it can neutralize the NF-
B-activating effect of the
GSE as well as the activation of NF-
B by TNF (Fig. 3B).
The activity of full-length SIRP
depends on the phosphorylation of
tyrosine residues in the cytoplasmic part of the protein by Src
tyrosine kinase (13); only the phosphorylated form of SIRP
can bind
SHP-1 and -2 protein phosphatases (13, 22). We checked whether this
phosphorylation is crucial to the NF-
B-inducing activity of the
cytoplasmic fragment of SHPS-1/SIRP
. In these experiments we used
the construct expressing the human homologue, named GSESIRP, of mouse
GSE2-1 that has a similar biological effect on NF-
B. Substitution
of tyrosine for phenylalanine within the consensus of immunoreceptor
tyrosine-based inhibitory motif (ITIM) (V/IXYXXV/L) decreased the ability of
GSE1FSIRP to activate NF-
B in all cell lines tested (Fig.
4). The mutation form of GSE with three
tyrosines changed for phenylalanines completely lost the ability to
activate NF-
B (data not shown). It is noteworthy that all the
versions of GSE2 and GSESIRP were expressed in our experiments at a
level comparable with those of endogenous SHPS-1/SIRP
, whereas ectopic expression of full-length constructs resulted in high levels of
SHPS-1/SIRP
that significantly exceeded the level of endogenous
protein (Fig. 3C).

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Fig. 4.
The substitution of ITIM tyrosine for
phenylalanine (GSE1FSIRP) decreases the ability of GSE to activate
NF- B-specific transcription. HeLa and NIH3T3 cells
were transfected and analyzed as described in the legend to Fig.
3A. All data represent the average of two experiments.
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The Cytoplasmic Domain of SHPS-1/SIRP
Is Likely to
Act through a Dominant Negative Mechanism--
Expression of the
cytoplasmic portion of SHPS-1/SIRP
protected cells against
TNF-induced apoptosis, presumably through its ability to activate
NF-
B. On the contrary, ectopic expression of full-length
SHPS-1/SIRP
makes cells more sensitive to TNF-stimulated apoptosis
(Fig. 5A). This effect was
stronger in HeLa than in NIH3T3 cells because of higher sensitivity of
NIH3T3 cells to TNF-specific apoptosis (Fig. 5A).
Consistently, full-length SHPS-1/SIRP
caused an inhibitory effect on
NF-
B activity (Fig. 3A). These observations suggested
that the cytoplasmic portion of SHPS-1/SIRP
acts as a dominant
negative mutant. In fact, cotransfection of cells with different ratios
of plasmids encoding truncated and full-length versions of
SHPS-1/SIRP
showed that the excess of full-length SHPS-1/SIRP
decreased the ability of GSE2-1 to activate NF-
B (Fig.
5B).

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Fig. 5.
GSE2-1 acts as a dominant negative form of
SIRP . A, overexpression of full-length SIRP
sensitizes cells to TNF. The indicated cells were treated with TNF
(0.01 ng/ml) and CHI (1 µg/ml for NIH3T3 and 5 µg/ml for HeLa) for
8 h. Remaining cells were quantitated by methylene blue assay.
Data represent the average of three experiments. B,
overexpression of full-length SIRP decreases activation of NF- B
by GSE2-1. Because of the high level of SIRP expression (see Fig.
3C), we used 50 and 100 ng of SIRP -expressing plasmid in
cotransfection with 1 µg of GSE2-1-expressing vector to reach
5-10-fold excess of SIRP over GSE2-1. In control transfection, 1 µg of pLXSN was used instead of 1 µg of GSE2-1-expressing vector.
Column 1, control 293 cells; column 2, GSE2-1;
column 3, SIRP /GSE2-1, 5:1; column 4,
pcDNA3/GSE2-1, 5:1; column 5, SIRP /GSE2-1, 10:1;
column 6, pcDNA3/GSE2-1, 10:1. The data represent the
average of two experiments.
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Activation of NF-
B by the Cytoplasmic Portion of
SHPS-1/SIRP
is Akt-dependent--
GSE2-1
did not stimulate NF-
B-dependent transcription in
serum-free medium in NIH3T3 cells (Fig.
6A), suggesting the
involvement of PI3K/Akt signaling in the GSE activity. We used
wortmannin, an inhibitor of PI3K kinase (26), to analyze whether
GSE2-1 indeed needs PI3K/Akt to activate NF-
B. Cells were
transfected and maintained in serum-free medium overnight. An
additional 10% of fetal calf serum activated NF-
B-mediated
transcription of the reporter, and the expression of GSE2-1 had an
additive effect (Fig. 6A). Wortmannin completely inhibited
NF-
B-mediated transcription caused both by addition of growth
factors and by GSE2-1 (Fig. 6A).

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Fig. 6.
GSE2-1-mediated activation of
NF- B is sensitive to wortmannin and depends on growth
factors, suggesting involvement of Akt. A, NIH3T3 cells
were transfected with the indicated plasmids and incubated overnight in
serum-free medium followed by addition of 10% fetal serum. Wortmannin
(100 nM) added 30 min before serum suppressed activation of
NF- B with both serum and GSE2-1. B, expression of
GSE2-1 does not protect Akt phosphorylation from inhibitory activity
of wortmannin in 293 cells. The results of Western immunoblotting with
anti-human phospho-Akt (pAkt) antibodies were as follows. Protein
extract from 293 cells are shown transfected with: insert-free vector,
cells maintained in serum-free medium, column 1; insert-free
vector after 30 min in 10% fetal serum, column 2;
GSE2-1-expressing vector after 30 min in 10% fetal serum,
column 3; GSE2-1-expressing vector, cells treated for 30 min with wortmannin (100 nM) before addition of serum,
column 4; insert-free vector, cells treated for 30 min with
0.01 ng of TNF in 10% fetal serum, column 5. Notice that
only 60-70% of 293 cells were transfected with GSE2-1-expressing
vector. The amounts of pAkt were quantitated and normalized to the
amount of actin.
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The inhibition of PI3K activity suppresses phosphorylation of Akt (26).
We analyzed the ability of GSE2-1 to regulate phosphorylation of Akt
using antibodies specific to the phosphorylated form of Akt. In 293 cells growing in serum-free medium overnight, the medium was changed to
serum-free every 30 min 4 times before protein extraction to avoid
contamination with 293 cells-secreted factors. The treatment of these
cells with 10% fetal serum for 30 min significantly increased Akt
phosphorylation (Fig. 6B). The GSE2-1 transiently transfected cells (60 to 70% of cells were transfected) in fetal serum
containing medium showed additional increase of Akt phosphorylation. Treatment of the cells with TNF had an effect on Akt phosphorylation similar to GSE2-1 expression (Fig. 6B). Expression of
GSE2-1 did not protect Akt phosphorylation from the inhibitory
activity of wortmannin (Fig. 6B).
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DISCUSSION |
In our search for GSEs protecting cells against TNF-induced
apoptosis, we isolated a genetic element encoding the cytoplasmic part
of an already known protein, SHPS-1/SIRP
. The isolated GSE protected
cells from TNF and strongly induced transcriptional activity of NF-
B
in a PI3K/Akt-dependent manner. Because NF-
B is known to
protect cells from TNF, GSE-mediated activation of this transcription
factor is presumably responsible for the inhibition of TNF-mediated
apoptosis. Full-length SHPS-1/SIRP
shows effects opposite to those
of the isolated GSE, suggesting that the GSE-encoded truncated form of
this protein acts through a dominant negative mechanism, interfering
with the function of the full-length protein.
What is the mechanism linking SHPS-1/SIRP
to NF-
B? The PI3K/Akt
pathway is important for NF-
B activation by TNF (26, 28). Activated
Akt phosphorylates the IKK
complex, which phosphorylates NF-
B inhibitor I
B and stimulates its degradation (29), which leads to a release of NF-
B that accumulates in the nucleus and activates transcription of the target genes. The functional connection of SIRP
with the PI3K/Akt pathway was shown. Overexpression of SIRP
, but not of its tyrosineless mutant form, inhibits PI3K activation and stimulation of proliferation by growth factors (13, 22).
On the contrary, ectopic expression of the cytoplasmic portion of
SHPS1/SIRP
stimulates growth factor-dependent Akt phosphorylation. NF-
B activation seems to be the result of this effect because it was completely suppressed by the PI3K inhibitor wortmannin. Importantly, this effect of the GSE-encoded truncated protein was also dependent on the presence of tyrosine residues. These
observations are consistent with the hypothetical model that the
truncated dominant negative form of SHPS1/SIRP
competes with the
full-length membrane-bound protein for some factors that are bound to
these proteins in a tyrosine phosphorylation-dependent manner. There are two proteins belonging to the same family that are
known to bind with SHPS1/SIRP
and mediate its biological functions,
tyrosine phosphatases SHP-1 and -2 (15). SHPS1/SIRP
can bind both
proteins when its own tyrosine residues in ITIM signals are
phosphorylated with Src (13, 14, 22).
SHP-1 and -2 are involved in many pathways, the function of which
requires tyrosine phosphorylation (30-32), including NF-
B signaling
(16, 33). Despite significant homology between SHP-1 and -2, these
proteins have opposite effects on cell viability. SHP-1 is
pro-apoptotic and can suppress cell growth (34-37), and its absence
stimulates NF-
B activation by TNF (33). In contrast, SHP-2 has an
anti-apoptotic effect (38), stimulating Akt activation by growth
factors (16, 39); its absence inhibits NF-
B activation by TNF (16).
NF-
B activity is increased in cells from the Mev strain of mice
deficient in SHP-1 (33). The opposite biological effect is associated
with SHP-2 gene deficiency (16).
SHP-1 is linked to NF-
B through modulation of PI3K/Akt signaling. It
causes dephosphorylation of tyrosine 688 inside the N-terminal SH2
domain of the regulatory subunit of PI3K p85 that turns it into an
inhibitor of the catalytic subunit p110 (40, 41). The depletion of
SHP-1 by the GSE-encoded cytoplasmic portion of SHPS-1/SIRP
might
lead to the activation of the p110 subunit of PI3K and phosphorylation
of Akt. Thus, dephosphorylation of the p85 regulatory subunit of PI3K
might be one of the mechanisms through which dominant negative
SHPS-1/SIRP
could activate NF-
B and promote TNF resistance.
However, in our experiments GSE2 caused only a slight increase in the
level of Akt phosphorylation in 293 cells, making it unlikely that this
effect is solely responsible for the dramatic activation of
NF-
B-dependent transcription. There could be additional
targets for GSE2 downstream of Akt that remain to be identified.
Although the function of SHP-1 is consistent with the observed
biological effects, the role of SHP-2 in our model remains less
defined. There are contradictory reports on the activity of this
phosphatase. There is evidence indicating that inactivation of SHP-2
decreases the transactivating abilities of NF-
B in response to
interleukin 1 (16). At the same time, other reports claim that
inactivation of SHP-2 increases the PI3K-dependent Akt
activation with EGF (42) and that cooperation of SIRP
and SHP-2 had
a negative effect on EGF-specific activation of PI3K (22). According to
these data, the functions of the two phosphatases are similar and
titration of SHP-2 by GSE2 could also stimulate
EGF/PI3K-dependent activation of NF-
B. This apparent
controversy has to be resolved in future experiments.
In summary, the biological effects of full-length SHPS-1/SIRP
can be
explained through its ability to bind and compartmentalize SHP-1, and
possibly SHP-2, with growth factor receptors causing their
inactivation. We hypothesize that GSE-encoded proteins act as
scavengers of SHP-1 and -2 in the cell, affecting proper targeting of
these phosphatases to plasma membrane and thereby interfering with
their inhibitory activity against growth factor-mediated signaling.