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INTRODUCTION |
Insulin binding to its receptor results in tyrosine
autophosphorylation of the
-subunit. This results in an active
insulin receptor that is able to phosphorylate several cytoplasmic
proteins on tyrosine residues. The tyrosine-phosphorylated insulin
receptor interacts with downstream docking proteins that appear to be
essential for insulin signaling. Phosphorylation of these adapter
proteins induces their association with proteins containing
SH21 domains, resulting in
activation of a variety of downstream signaling pathways. Examples of
such adapter proteins include the insulin receptor substrates (IRS) 1, 2, 3, and 4 and the SHC proto-oncogene product. These adapter proteins
appear to link the insulin receptor to at least two signaling pathways,
the phosphatidylinositol 3-kinase (PI3K) pathway and the
Ras-mitogen-activated protein kinase pathway. It remains unclear
whether these adapters constitute the full complement of signaling
intermediates utilized by the insulin receptor. In fact, much recent
evidence has begun to accumulate to suggest that the known signaling
pathways that emanate from the IR may be insufficient to fully explain
insulin-mediated metabolic regulation, and in particular the regulation
of GLUT4 translocation.
One of the most important signaling pathways that appears to be
required for many metabolic effects of insulin is the activation of
PI3K. Binding of the p85 regulatory subunit of PI3K to
tyrosine-phosphorylated IRS proteins leads to activation of the lipid
kinase activity of the p110 catalytic subunit of this enzyme (1, 2).
The metabolic effects of insulin that require PI3K activity include GLUT4 translocation, glucose uptake, activation of fatty acid synthase
and glycogen synthase, and stimulation of amino acid transport and
protein synthesis (3-5). All of these actions were inhibited either by
pharmacological inhibitors of PI3K or by expression of dominant
negative mutants of PI3K (3, 6, 7). For example, introduction of a
dominant negative form of the p85 subunit that cannot interact with the
p110 catalytic subunit completely inhibited activation of PI3K in
3T3-L1 adipocytes in response to insulin. This prevented GLUT4
translocation, suggesting that PI3K activity was necessary for GLUT4
translocation (8-10). This block was successfully reversed by the
co-expression of a constitutively active p110 subunit, which by itself
possessed the ability to promote GLUT4 translocation in transiently
transfected primary rat adipocytes and suggested that PI3K activity may
be sufficient for GLUT4 translocation (11-13).
Several downstream targets of PI3K such as PKB (also termed AKT) and
p70S6 kinase may play important roles in mediating
PI3K-dependent metabolic signaling. Stimulation of the
serine/threonine protein kinase PKB required the prior activation of
PI3K, due in part to a direct interaction of the PKB PH domain with the
secondary lipid messenger phosphatidylinositol 3,4,5-phosphate
(PIP3), which is believed to cause a conformational change
that allows subsequent phosphorylation of PKB by multiple upstream
kinases. In this regard, PKB becomes phosphorylated upon threonine 308 and serine 473 in response to insulin stimulation (14). One of these
upstream kinases, PIP3-dependent kinase 1 (PDK1), has been cloned and characterized and shown to phosphorylate
threonine 308 and thus activate PKB (15, 16). The PDK1 kinase also
contains a PH domain that is believed to mediate its interaction with
the plasma membrane in a phosphoinositide-dependent manner.
Studies in which a constitutively active PKB was targeted to the plasma
membrane using the Src myristoylation signal have demonstrated that, in
the absence of insulin stimulation, glucose uptake and GLUT4
translocation were enhanced (17, 18). These data support a role for
PI3K and PKB in the regulation of GLUT4 translocation.
However, whether activation of either PI3K or PKB is sufficient for the
maximal stimulation of GLUT4 translocation by insulin remains
controversial. Studies designed to address these questions have yielded
contradictory results. In a series of studies, adenoviral-mediated expression of either a constitutively activated form of p110 or a form
of p110 that was targeted to GLUT4 vesicles in 3T3-L1 adipocytes was
analyzed. These manipulations resulted in PI3K activities that exceeded
insulin-stimulated levels and increased GLUT4 translocation and glucose
transport, yet these effects were significantly reduced when compared
with insulin (19, 20). In other experiments, PI3K was activated in
3T3-L1 adipocytes using thiophosphorylated peptides corresponding to
the binding motif of PI3K within IRS1; again, only a modest increase in
GLUT4 translocation was observed (21). Other studies have shown that,
although growth factors such as PDGF can activate PI3K activity in
3T3-L1 adipocytes, this is not sufficient to stimulate significant
GLUT4 translocation to the plasma membrane (22, 23). In other
experiments, interleukin-4 stimulation of L6 myoblasts overexpressing
the interleukin-4 receptor induced IRS1 tyrosine phosphorylation and
increased PI3K activity to the same degree as insulin but had no effect
upon glucose uptake (22). More recently, a mutant PKB protein in which
the regulatory phosphorylation sites were substituted with alanine was
shown to exert a dominant negative effect on endogenous PKB activity stimulated by insulin. Expression of this dominant negative PKB in
3T3-L1 adipocytes had no effect on insulin-induced glucose uptake or
GLUT4 translocation but was found to decrease the rate of protein
synthesis and inhibit the ability of insulin to activate p70S6 kinase
(24). These data strongly suggest that PKB may not be necessary for
insulin-dependent GLUT4 translocation. More recently it was
found that introduction of membrane-permeant esters of PIP3
into 3T3-L1 adipocytes by themselves did not stimulate glucose uptake
or GLUT4 translocation to the plasma membrane. However, these esters
were able to reverse the wortmannin blockade of insulin-stimulated
GLUT4 translocation (25). This suggests that PIP3 is
necessary but not sufficient and raises the possibility that additional
signaling pathways may originate from the insulin receptor to mediate
the full response. In other experiments, microinjection of competitive
inhibitory IRS1 proteins, which effectively blocked the interaction of
the IR with endogenous IRS-1, had no effect upon GLUT4 translocation or
glucose uptake in response to insulin (8). These same proteins blocked
insulin-dependent membrane ruffling and mitogenesis,
suggesting that IRS-1 was critical for some insulin actions but not for
GLUT4 translocation. In summary, the current data strongly support an
essential role for PI3K in the regulation of glucose uptake by insulin.
However, the data from a variety of studies outlined above suggest the
possibility that other signaling pathways may exist that emanate
specifically from the insulin receptor that are responsible for the
full metabolic response of a muscle or adipose cell to insulin.
In this paper we report the cloning of mouse APS, which was shown to
interact strongly with the insulin receptor. APS mRNA and protein
were expressed predominantly in skeletal muscle, heart, fat, and 3T3-L1
adipocytes. The SH2 domain of APS interacted with the activation loop
of the kinase domain of the insulin receptor. Endogenous APS becomes
robustly tyrosine-phosphorylated on tyrosine 618 after insulin
treatment of 3T3-L1 adipocytes. These results are suggestive of a
physiological role for APS in insulin signaling in the
insulin-responsive tissues muscle and fat.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
CHO-IR cells overexpressing the human insulin
receptor (26) were grown in F12 nutrient medium containing 10% fetal
bovine serum and antibiotics. 3T3-L1 fibroblasts were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and antibiotics. Differentiation to adipocytes was induced
as described previously (9). The cells were then cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and antibiotics for 8-12 days. Before growth factor
stimulation the cells were serum-starved for 2-18 h. Unless otherwise
indicated, 100 nM insulin was added directly to the medium
and the incubation was continued at 37 °C for the indicated times.
Plasmid Constructions--
Full-length and subdomains of APS
were subcloned into either the two-hybrid plasmid pEG202, the mammalian
expression plasmid, pcDNA3.1His (Invitrogen), pGEX5X (Amersham
Pharmacia Biotech), or pET28 (Novagen) using standard methods. All
site-directed mutants were generated by QuikChange kit (Stratagene).
Yeast Strains and Plasmids--
S. cerevisiae EGY48
(a-trp1, ura3-52, his3, leu2) and all expression plasmids
were provided by the laboratory of Roger Brent and have been described
previously (9, 27-31). All procedures for routine growth and
maintenance of yeast strains have been described previously. Plasmid
transformation of yeast was by the lithium acetate method (32). The
insulin receptor and insulin-like growth factor 1 receptor cDNA
fusions have been reported previously (30, 31). The colony color
-galactosidase assay was performed as described (30). A 3T3-L1
adipocyte cDNA was generated with the ZAP cDNA synthesis kit
(Stratagene) and subcloned in the pJG4-5 library vector.
Northern Blots--
Northern blot analysis was performed on
commercial human and rat multiple tissue poly(A)+ RNA blots
(CLONTECH), or, purified poly(A)+ RNA
prepared from either 3T3-L1 fibroblasts or adipocytes. For the Northern
blots shown in Fig. 2, probes were generated by polymerase chain
reaction and labeled by nick translation. Hybridization was performed
overnight at 60 °C in 15% formamide, 1% bovine serum albumin, 0.5 M sodium phosphate (pH 7.2), 7% SDS, 100 µM
EDTA, and 50 µg ml
1 denatured salmon sperm DNA with
high stringency washes.
In Vitro Interaction Studies--
A GST fusion protein was
generated by introducing the APS cDNA fragment corresponding to the
SH2 domain (amino acids 408-507) into the pGEX5X expression plasmid.
After transformation of DH5
, induction with 1 mM
isopropyl-1-thio-
-D-galactopyranoside, cell collection,
and lysis by sonication, the proteins were purified using immobilized
glutathione-agarose beads. Serum-starved cultured CHO-IR cells were
stimulated with 100 nM insulin for 0, 5, 15, or 30 min at
37 °C, washed twice with ice-cold 1× PBS, and solubilized with
lysis buffer (1× PBS supplemented with 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM sodium vanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate). The samples were homogenized and
clarified by centrifugation, and incubated (500 µg of total
protein/reaction) for 2 h at 4 °C with 3-5 µg of immobilized
GST fusion protein. After extensive washing with ice-cold HNTG buffer
(10 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol), the proteins co-associating with the GST fusion
proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane (Amersham Pharmacia Biotech), and immunoblotted as described below with either anti-phosphotyrosine antibody 4G10 or antibodies against the
subunit of the insulin receptor.
Transient Transfection of CHO-IR Cells--
CHO-IR cells were
transiently transfected with 5 µg of the various APS constructs
subcloned in the mammalian expression vector pcDNA3.1His
(Invitrogen) by LipofectAMINE according to manufacturer's instructions
(Life Technologies, Inc.). After 48 h, the cells were
serum-starved overnight and then stimulated by the addition of 100 nM insulin for 5 min. Lysates were prepared as described under "Immunoprecipitation and Immunoblots."
Immunoprecipitation and Immunoblots--
Cells treated as
described under "Results and Discussion" were washed twice with
ice-cold 1× PBS and then lysed in 1 ml of lysis buffer. After a 30-min
centrifugation at 4 °C to remove the insoluble material, proteins of
interest were immunoprecipitated with either mouse monoclonal
antibodies or rabbit polyclonal antibodies coupled to protein
A/G-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) during a
2-3-h incubation at 4 °C with constant mixing. The immune complexes
were washed three times with ice-cold HNTG buffer. Proteins were
separated by SDS-polyacrylamide gel electrophoresis and transferred to
PVDF membrane (Amersham Pharmacia Biotech). Membranes were incubated
for 1 h at room temperature in TBST buffer (Tris·HCl, pH 7.4, NaCl, 0.01% Tween 40) containing 5% nonfat dry milk and blotted with
specific antibodies in TBST containing 5% bovine serum albumin for 2 to 3 h. Immunoreactive bands were detected by ECL immunodetection
system (Pierce).
Antibodies--
Anti-phosphotyrosine antibody 4G10 was purchased
from Upstate Biotechnology (Lake Placid, NY), anti-insulin receptor
antibody was purchased from Santa Cruz Biotechnology, and anti-XPRESS
mouse monoclonal was purchased from Invitrogen.
Fusion protein was produced in Escherichia coli from a
pGEX5X (Amersham Pharmacia Biotech) expression plasmid containing the COOH domain (amino acids 508-591) of APS. The GST fusion protein was
purified by standard procedures. Immunizing rabbits with the purified
GST fusion raised anti-APS antisera.
Protein Determination and Gel Analysis--
Protein assays were
performed by modified Lowry method (33). SDS-polyacrylamide gel
electrophoresis was performed as described by Laemmli (34).
 |
RESULTS AND DISCUSSION |
Identification of APS as an Insulin Receptor Interactor Using the
Yeast Two-hybrid System--
To be able to identify new substrates of
the insulin receptor that may be important components in metabolic
signaling pathways, we carried out a yeast two-hybrid screen with the
cytoplasmic domain of the human insulin receptor as bait and searched
for interacting proteins within a 3T3-L1 adipocyte cDNA library. Of 4 × 106 transformants, 90 colonies showed specific
interaction with the insulin receptor. Sequence analysis of the rescued
plasmids revealed that 17 encoded a recently identified adapter protein
termed APS (adapter protein containing PH and SH2 domain). The sequence
of the longest clone contained an insert of 2832 base pairs. Analysis of potential coding sequences suggested a protein of 621 amino acids
with a predicted molecular weight of 66,553. Analysis of the predicted
amino acid sequence revealed a PH domain encoded by amino acids
197-299 and an SH2 domain between amino acids 408 and 507. Two
potential tyrosine phosphorylation sites were located at amino acids 46 (NPXY) and 618 (ENQY). Alignment of the mouse APS amino acid
sequence with the published human and a partial rat cDNA (that we
also cloned in an insulin receptor two-hybrid screen with a skeletal
muscle library) revealed that there was significant conservation
between species (Fig. 1). The only marked difference between these proteins was within the amino-terminal domain
where two short stretches are missing from the mouse: amino acids 12- 16 (APVPV), amino acids 84-89 (GPTTRG) of the human APS, and one short
stretch missing from the human: amino acids 161-164 (PASE) of mouse
APS.

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Fig. 1.
Amino acid alignment between human
(h), mouse (m), and partial rat
(r) sequence of APS with the structural domains
highlighted as described: PH domain ( ), SH2 domain ( ), and
potential tyrosine phosphorylation sites ( ).
Asterisk (*) indicates conserved amino acids between all
species.
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APS is a recently described protein whose function is unknown. APS was
first described to interact with an oncogenic mutant of the tyrosine
kinase receptor, c-Kit (35). Activation of immunoreceptor signaling
induced tyrosine phosphorylation of APS in response to a variety of
cytokines including stem cell factor. As reported previously, APS is
similar to but not identical to SH2B and LNK (35). The PH domains of
SH2B and LNK are 58% and 40% identical to APS, whereas the SH2
domains of SH2B and LNK are more similar with 80% and 72% identity,
respectively. The LNK protein that has been reported may not represent
the complete cDNA or may be a result of alternative splicing or
start codon usage. Comparison of the remaining sequences of the three
proteins outside either the PH or SH2 domain showed little sequence
similarity. However, one of the potential tyrosine phosphorylation
sites at the carboxyl terminus of APS, tyrosine 618, is conserved in
both SH2B (amino acid 753) and LNK (amino acid 297). SH2B was
originally cloned from mast cells because of its ability to bind in a
yeast tribrid system to the tyrosyl-phosphorylated
subunit of the
high affinity immunoglobulin E (IgE) receptor (36). SH2B has also been
reported to bind to tyrosine-phosphorylated JAK2 upon growth hormone
stimulation of 3T3-F442A adipocytes (37). These data suggest that
growth hormone activation of JAK2 recruits SH2B, thereby initiating an as-yet-unidentified signal transduction pathway. The second family member, LNK, is tyrosine-phosphorylated upon T-lymphocyte activation by
antibody-mediated cross-linking of the T cell receptor and CD4. LNK has
been suggested to provide a link between the activated T cell receptor
and phosphatidylinositol 3-kinase, phospholipase C
1, and Ras
signaling pathways through a multifunctional tyrosine phosphorylation
site within the protein (38, 39). Thus, APS, SH2B, and LNK appear to be
a family of adapter molecules involved in tyrosine kinase signaling in
hematopoietic cells.
Expression of APS in Tissues and Cell Lines--
To determine the
tissue distribution of APS mRNA, both human and rat multiple tissue
Northern blots were hybridized with a probe generated against the SH2
domain of APS. Two APS mRNA transcripts were detected with tissue
specificity and variation in their relative abundance (Fig.
2A). The transcripts were approximately 1.9 and 2.9 kbp in
size. Both were expressed with equal intensity in poly(A)+
mRNA prepared from either rat or human skeletal muscle. However, the lower RNA band of approximate 1.9 kbp was absent in other tissues
examined. The significance of the multiple mRNA bands remains to be determined.
Since the full-length APS cDNA was isolated from a 3T3-L1 adipocyte
cDNA library, we examined the expression of APS mRNA in 3T3-L1
cells. Northern blot analysis of poly(A)+ RNA isolated from
fibroblasts and fully differentiated adipocytes was performed with a
cDNA probe encoding the SH2 domain of APS. As shown in Fig.
2B, a transcript of 2.9 kbp
was observed in RNA prepared from both fibroblasts and adipocytes. This
corresponds well to the size of the full-length cDNAs that we
identified in the two-hybrid assay, the longest of which was 2833 base
pairs. APS mRNA expression was highest in the adipocytes. The blot
was stripped and rehybridized with a GLUT4 probe. In agreement with published data, GLUT4 expression is up-regulated in response to differentiation of the fibroblasts.

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Fig. 2.
Tissue distribution of APS in rat and human
tissues. A, multiple tissue Northern blots derived from
a variety of human or rat tissues were hybridized with an APS SH2
domain probe. Lanes 1-8 and lanes
9-16 are prepared from either rat or human tissue samples,
respectively. Lanes 1 and 9, heart;
lanes 2 and 10, brain; lane
3, spleen; lanes 4 and 12,
lung; lanes 5 and 13, liver;
lanes 6 and 14, skeletal muscle;
lanes 7 and 15, kidney;
lane 8, testis; lane 11,
placenta; lane 16, pancreas. B,
expression of APS in 3T3-L1 fibroblasts and in differentiated
adipocytes. A Northern blot containing poly(A)+ RNA from
3T3-L1 fibroblasts (lane 1) and differentiated
adipocytes (lane 2) was hybridized with the APS
SH2 domain probe. The bottom panel shows the
result of hybridization with a GLUT4 probe. The migration of the RNA
size markers (in kbp) is shown on the left, and the
arrows on the right denote the position of the
APS and GLUT4 RNA transcripts.
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In Vitro Interaction of the APS SH2 Domain with the Insulin
Receptor--
To further examine the interaction of APS and the
insulin receptor, we produced a GST fusion protein that contained the
SH2 domain of APS (amino acids 408-507). We examined the ability of this GST fusion protein to interact with proteins in lysates derived from unstimulated or insulin-stimulated CHO cells that overexpress the
insulin receptor. After incubation of the immobilized GST protein with
cellular extracts, the samples were extensively washed and the
coprecipitating proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and immunoblotted with an anti-insulin receptor antibody (Fig. 3A,
upper panel). The blots were then stripped and
reblotted with an anti-phosphotyrosine antibody (Fig. 3A, lower panel). GST-SH2 APS fusion protein
precipitated the insulin receptor from these lysates in a
time-dependent manner. The coprecipitation was dependent
upon prior stimulation of the insulin receptor, as shown by the lack of
association between these two proteins in unstimulated lysates.

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Fig. 3.
In vitro interaction of
the APS SH2 domain with insulin receptor. GST fusion proteins
corresponding to the SH2 domain of APS, SH2B, and LNK were assayed for
the ability to interact in vitro with the IR expressed in
CHO-IR cells or 3T3-L1 adipocytes. A, cellular extracts (500 µg) were prepared from CHO-IR cells, stimulated with or without
insulin (100 nM) for the times indicated and incubated with
3 µg of GST-SH2 fusion protein of APS. Samples were resolved by 8%
SDS-polyacrylamide gel electrophoresis, transferred to PVDF and
immunoblotted (IB) with anti-insulin receptor antibodies
(IR), and then stripped and reprobed with
anti-phosphotyrosine antibodies (PY). An arrow on
the right denotes the subunit of the insulin receptor.
B, cellular extracts (500µg) were prepared from 3T3-L1
adipocytes, stimulated with or without insulin (100 nM) for
the times indicated, and incubated with 5µg of GST-SH2 fusion
proteins of APS (lane 1), SH2B (lane
2), or LNK (lane 3). Samples were
immunoblotted (IB) with anti-insulin receptor antibodies
(IR). 50 µg of total cellular extracts (lane
4) were run as a control. An arrow on the
right denotes the subunit of the insulin receptor.
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Recent work has suggested that the SH2 domain of SH2B, an APS-related
protein, was able to interact with the insulin receptor (40, 41). We
therefore produced GST fusion proteins that contained the SH2 domains
of APS, SH2B, or LNK and directly compared the ability of these GST
fusion proteins to interact with the cytoplasmic domain of the insulin
receptor in lysates derived from unstimulated or insulin-stimulated
3T3-L1 adipocytes (Fig. 3B). As shown previously, the
GST-SH2 APS fusion protein was found to bind the insulin receptor in an
insulin-dependent manner. Using equivalent amounts of GST fusion (5 µg), neither the GST-SH2 SH2B or GST-SH2 LNK was able to
interact with the receptor to the same degree as the SH2 domain of APS.
This difference between our results and the previous studies may be due
to the higher amounts of GST fusion protein used in their study, a
5-fold higher amount than that shown in Fig. 3B. This is in
agreement with data from our laboratory in experiments where higher
amounts of GST fusion proteins were used (10 µg), which revealed a
weak interaction between both SH2B and LNK with the insulin receptor
(data not shown).
Characterization of the Site of Interaction between APS and the
Insulin Receptor--
To further analyze insulin receptor binding to
APS, we mapped the site of interaction between these two proteins using
a detailed two-hybrid analysis with a variety of insulin and the
insulin-like growth factor receptor mutants. We detected strong
interaction between APS and wild-type insulin receptor and insulin-like
growth factor receptor as determined by the intensity of the blue color in the colony color assay. This interaction was dependent upon receptor
activity since mutation of the critical lysine (to alanine) within the
ATP binding sites of both receptors eliminated all activity. The
cytoplasmic domain of the insulin receptor contains several tyrosine
residues that, when phosphorylated, create docking sites for multiple
downstream adapter proteins including SHC and IRS-1, which requires
tyrosine 960 for interaction (35, 36), and GRB10, which appears to
interact with the activation loop tyrosines (residues 1146, 1150, and
1151) (42, 43). Substitutions of these tyrosine residues with
phenylalanine enabled us to map the site of interaction between APS and
the insulin receptor. We found that the following tyrosine residues
were not necessary for APS interaction: 953, 960, 1316, and 1322. However, a double or single amino acid substitution of the tyrosine
residues within the autoregulatory loop, Y1150F/Y1151F or Y1146F,
greatly reduced the ability of these two proteins to interact. Deletion
of the 30-carboxyl-terminal amino acids of the insulin receptor also had no effect upon APS interaction (data not shown).
The fact that the interaction of APS with the insulin receptor required
tyrosine phosphorylation of the insulin receptor suggested that the SH2
domain within APS was the domain that bound the autoactivation loop of
the insulin receptor. Although this interaction was probably direct, it
remained possible that additional region(s) of APS were also important.
To investigate whether the SH2 domain of APS was critical, we
substituted the conserved arginine residue within the FLVR motif of the
SH2 domain to lysine (R437K) and studied the effect on the interaction
between APS and insulin receptor in the yeast two-hybrid system. This
arginine (R) to lysine (K) mutation in the FLVR motif has been shown
previously to lead to the loss in the ability of SH2 domains to bind to
phosphotyrosine. As shown in Fig. 4, the
wild-type full-length APS interacted strongly with the insulin receptor
as determined by colony color studies. We found that the point mutation
within the SH2 domain (R437K) of the full-length APS almost completely
abrogated the binding of APS to insulin receptor. Point mutations
within either the PH domain (W290L), which would be predicted to
destroy lipid binding, or the putative tyrosine phosphorylation site
(Y618F) had no effect on the interaction. Furthermore, a series of APS
hybrids were generated and tested for insulin receptor interaction
using the two-hybrid assay: the amino-terminal domain (amino acids
1-196), the PH domain (197-299), the SH2 domain (408-507), the
carboxyl-terminal domain (508-621), the SH2 domain plus the
carboxyl-terminal domain lacking the tyrosine phosphorylation site
(408-591), and the SH2 domain plus the full carboxyl terminus
(408-621). Only the proteins that contained the intact SH2 domain were
able to interact efficiently with the insulin receptor (Fig. 4). These
results suggest that the SH2 domain alone is sufficient for
interaction.

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Fig. 4.
Two-hybrid insulin receptor-APS
interaction. Full-length and various domains of APS were subcloned
in the activation domain plasmid pJG4-5 and assayed for interaction
with the wild-type insulin receptor (WT IR) in the yeast
two-hybrid system. Transformants were assayed by colony color, and the
data represent an average from a minimum of three independent colonies.
++++ indicates strong interaction (dark blue), + indicates weak
interaction (faint blue), and - refers to no interaction
(white).
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Antibodies and Tissue Expression--
In order to be able to
characterize the endogenous APS protein, rabbit polyclonal antibodies
were raised against the carboxyl-terminal (amino acids 507-591)
domains of APS. Lysates prepared from 3T3-L1 adipocytes were
immunoprecipitated with the anti-APS antibodies. The immunoprecipitates
were separated by SDS-polyacrylamide gel electrophoresis and
immunoblotted. Proteins with a molecular mass of between 60 and 70 kDa
were detected in 3T3-L1 adipocyte lysates with a major band at
approximately 70 kDa (Fig.
5A). These protein bands were
not observed in immunoprecipitates with any of the preimmune sera
tested (data not shown). The multiple bands may represent degraded
protein or alternatively spliced variants of APS expressed in 3T3-L1
adipocytes. In agreement with the Northern blot data, which showed
induction of APS mRNA levels in differentiated 3T3-L1 adipocytes
(Fig. 2B), APS protein was only detected in adipocytes (Fig.
5A). The fact that we observed no protein in fibroblasts
despite expression of detectable mRNA may be due to different
sensitivity of the blotting procedures or, alternatively, may be
indicative of post-transcriptional regulation. We next performed
immunoblot analysis of several human and rat tissues. APS protein was
specifically detected in human skeletal muscle and heart (Fig.
5B). No APS protein was detected in extracts prepared from
either human spleen or liver. Interestingly, the human APS was slightly
reduced in molecular mass with an approximate molecular mass between 60 and 65 kDa compared to the protein detected in the mouse 3T3-L1
adipocyte cell line. The origin of these proteins of distinct
mobilities remains unclear. Immunoblot analysis of adult rat tissues
clearly demonstrated that the APS protein was expressed in a variety of
insulin responsive tissues including adipose, heart, and pancreas (Fig.
5C). APS protein was also detected in rat skeletal muscle
with a molecular mass similar to that found for human APS (data not
shown). The apparently high level of APS protein in pancreas and the
low level observed in spleen would not be predicted by the Northern
blot data, which is otherwise consistent. Nevertheless, the
demonstration that APS is expressed primarily in insulin-responsive
tissues and in 3T3-L1 adipocytes supports a potential physiological
role of this protein in insulin signaling.

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Fig. 5.
Expression of APS protein in human
and mouse tissues. A, total cellular extracts were
prepared from either 3T3-L1 fibroblasts (lane 1)
or fully differentiated adipocytes (lane 2). 2 mg
of total protein were immunoprecipitated with anti-APS antibodies as
described under "Experimental Procedures." Samples were resolved on
an 8% SDS-polyacrylamide gel, transferred to PVDF, and immunoblotted.
APS is denoted by arrows on the right, and
standard molecular mass markers (kDa) are shown on left.
B, human tissue extracts were purchased from
CLONTECH. 5 mg of total protein from human skeletal
muscle (lane 2), human heart (lane
3), human liver (lane 4), and human
spleen (lane 5) or 500 µg of total protein from
mouse 3T3-L1 adipocytes (lane 1) were
immunoprecipitated with anti-APS antibodies and immunoblotted with
anti-APS antibodies. C, rat tissue extracts were prepared
from either adipose (lanes 1 and 2),
heart (lanes 3 and 4), pancreas
(lanes 5 and 6) or spleen
(lanes 7 and 8). 10 mg of total
protein were immunoprecipitated with either pre-immune
(lanes 1, 3, 5, and
7) or anti-APS (lanes 2, 4,
6, and 8) antibodies as described under
"Experimental Procedures." Samples were immunoblotted with anti-APS
antibodies. Arrows on the right denote APS with
standard molecular size markers (kDa) shown between lanes
2 and 3.
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Endogenous APS Is Tyrosine-phosphorylated by Insulin Stimulation of
3T3-L1 Adipocytes--
It is clear from the GST pull-down experiments
that the SH2 domain of APS interacts with the insulin receptor in a
phosphotyrosine-dependent manner. Furthermore, the results
from the yeast two-hybrid experiments indicate that APS possesses the
ability to interact directly with insulin receptor. To gain further
insight regarding the functional importance of the insulin receptor and
APS association, we examined the effects of insulin or PDGF treatment
on tyrosine phosphorylation on endogenous APS in 3T3-L1 adipocytes.
3T3-L1 lysates derived from cells stimulated with either insulin or
PDGF were immunoprecipitated with anti-APS antibodies and blotted with
either anti-phosphotyrosine (Fig.
6A, upper
panel) or anti-APS antibodies (Fig. 6A,
lower panel). Insulin induced a rapid but
transient tyrosine phosphorylation of APS in 3T3-L1 adipocytes.
Phosphotyrosine content of APS was highest after 5 min of insulin
stimulation and was slightly reduced at 15 min. It is clear from these
results that APS is an endogenous substrate of the insulin receptor.
Immunoblotting with the anti-APS antibodies indicated that equivalent
amounts of APS had been immunoprecipitated under all conditions
examined. APS was not tyrosine-phosphorylated in response to PDGF
stimulation of 3T3-L1 adipocytes, even though the receptor was clearly
shown to be tyrosine-phosphorylated, suggesting some degree of receptor
specificity (Fig. 6B). Despite our demonstration that APS is
a substrate of the IR, we have thus far been unable to demonstrate a
stable interaction between the IR and APS by co-immunoprecipitation
(data not shown). In this regard, it should be noted that
co-immunoprecipitation of other substrates of the IR, including SHC and
IRS proteins, has also been difficult to demonstrate. Our data do not
allow us to compare the relative levels of APS to other known
substrates of the IR such as IRS-1, IRS-2, or SHC since we do not know
the relative efficiencies of immunoprecipitation or immunoblotting of
the various antibodies. The fact that we cannot easily see a
tyrosine-phosphorylated band in total cell lysates from 3T3-L1 cells or
tissues suggests that that APS may not be as abundant as IRS-1.
Alternatively, this may be partly due to the finding that APS is
phosphorylated upon only a single site, whereas IRS-1 is multiply
phosphorylated. In this regard, the SHC protein, which is a known
substrate of the IR that is phosphorylated upon a single tyrosine, is
also not easily observed in insulin-stimulated lysates without prior immunoprecipitation.

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Fig. 6.
Tyrosine phosphorylation of endogenous APS in
3T3-L1 cells. A, 500 µg of total cellular extract
prepared from 3T3-L1 adipocytes, stimulated with or without insulin
(100 nM, lanes 1-3) or PDGF-
(50 ng/ml, lanes 4-6) for the times indicated.
The samples were immunoprecipitated with anti-APS antibodies and
immunoblotted (IB) with anti-phosphotyrosine antibodies
(PY) or with anti-APS antibodies (APS).
B, 50 µg of total cellular extracts prepared from
unstimulated (lane 1), 5 min (lane
2) or 15 min (lane 3)
insulin-stimulated or 5 min (lane 4) or 15 min
(lane 5) PDGF -stimulated 3T3-L1 adipocytes
were immunoblotted (IB) with anti-phosphotyrosine antibodies
(PY). Arrows on the right denote the
position of tyrosine phosphorylated insulin receptor (IR)
and the PDGF receptor (PDGFR) with standard molecular size
markers (kDa) shown on the left.
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Unlike activation of PI3K and mitogen-activated protein kinase, which
can both become activated to varying degrees by insulin and PDGF, APS
appears to be a specific substrate of the insulin receptor. In
agreement with our studies showing limited in vitro interaction between the insulin receptor and the APS-related protein SH2B, no tyrosine phosphorylation of SH2B has been reported in response
to insulin stimulation either in CHO cells overexpressing the insulin
receptor (40, 41) or in 3T3-F442A adipocytes (44). More recently
SH2B
has been shown to be recruited and tyrosine-phosphorylated via
a direct interaction to the PDGF receptor. Co-immunoprecipitation of
endogenous SH2B
and the PDGF receptor was detected after ligand stimulation in both NIH3T3 and 3T3-F442A adipocytes (44). We have been
unable so far to detect either a stable association between APS and the
PDGF receptor (data not shown) or to detect tyrosine phosphorylation of
APS by PDGF
in 3T3-L1 adipocytes. This appears to be one more
difference between the members of this family of adapter proteins.
The SH2 Domain and Tyrosine 618 Are Essential for APS
Phosphorylation--
In order to be able to characterize the relative
importance of the SH2 domain, the PH domain and the carboxyl-terminal
tyrosine residue in the tyrosine phosphorylation of APS by the insulin receptor, plasmids containing the full-length wild-type and mutant forms of APS with an XPRESS epitope tag at the amino terminus were
transiently transfected into CHO-IR cells. We compared the phosphotyrosine content between wild-type APS and full-length APS
containing single point mutations within either the PH domain (W290L),
the SH2 domain (R437K), or a potential tyrosine phosphorylation site
(Y618F). Lysates prepared from the transfected cells were immunoprecipitated with anti-APS antibodies. The immunoprecipitates were immunoblotted with anti-phosphotyrosine (Fig.
7, upper panel). The blots were then stripped and reblotted with the epitope tag antibody, anti-XPRESS (Fig. 7, lower panel). The
transfected wild-type APS protein was found to undergo heavy tyrosine
phosphorylation upon the addition of 100 nM insulin for 5 min. Mutation of the conserved tryptophan to leucine (W290L) in the PH
domain greatly reduced, but did not eliminate the ability of APS to
become tyrosine-phosphorylated in response to insulin. Expression of
the APS mutant in which the conserved arginine in the FLVRES motif in
the SH2 domain was mutated to lysine (R437K) abolished all tyrosine
phosphorylation of APS in response to insulin stimulation. Similarly,
mutation of tyrosine 618 completely abolished the tyrosine
phosphorylation of APS.

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Fig. 7.
Transient transfection of CHO-IR cells with
no DNA (NO), wild-type APS (WT),
W292L APS (PH*), R439K APS (SH2*),
and Y618F APS (Y*), performed as described under
"Experimental Procedures." Cellular extracts were prepared
from these transfected CHO cells, stimulated with or without insulin
(100 nM) for the times indicated (min). 2 mg of cellular
extract were immunoprecipitated with anti-APS antibodies and
immunoblotted with anti-phosphotyrosine antibodies (PY). The
blot was then stripped and reprobed with anti-XPRESS antibodies
(XPRESS). An arrow on the right
denotes the position of APS. The standard molecular size markers (kDa)
are shown on the left.
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These results show that a functional SH2 domain is critical for
tyrosine phosphorylation of APS in response to insulin and is in
agreement with the studies mapping the site of interaction between APS
and the insulin receptor in yeast. PH domains are found in a large
variety of proteins and are likely to be involved in the localization
of proteins to the proximity of the membrane. Transient transfection of
CHO cells overexpressing the insulin receptor with APS containing a
mutation of the conserved tryptophan greatly reduced the phosphotyrosyl
content of APS in response to insulin. Although detailed analysis of
the binding affinity of phospholipids has not been undertaken, the PH
domain may be important in regulating the intracellular localization of
APS and its recruitment to the receptor after insulin stimulation. Identification of the phosphorylation site within APS was clearly demonstrated to be the carboxyl-terminal tyrosine (position 618) since
mutation of this residue abolished all tyrosine phosphorylation of APS
in response to insulin. Immunoblotting with the epitope tag anti-XPRESS
antibody indicated equivalent amounts of transiently expressed APS had
been immunoprecipitated under all conditions. Transfection of CHO cells
with these plasmid cDNAs produced two proteins, of which only the
higher molecular weight form was able to be
tyrosine-phosphorylated.
As discussed earlier, many recent studies have suggested the existence
of additional signaling pathways which emanate from the insulin
receptor that are necessary for the full activation of glucose
transport by insulin. These unidentified signaling proteins could
interact specifically with the insulin receptor and in collaboration
with PI3K produce the full metabolic response within a muscle or
adipose cell. This report identifies a new substrate of the insulin
receptor that may play such a role. APS was identified through a yeast
two-hybrid screen using the insulin receptor as bait. The APS protein
is highly expressed in several insulin-responsive tissues. Endogenous
APS was found to be a direct substrate of the insulin receptor and
underwent tyrosine phosphorylation in response to insulin in 3T3-L1
adipocytes. We have also demonstrated a clear difference in APS
tyrosine phosphorylation by insulin and PDGF. The function of tyrosine
phosphorylation of APS in mediating the effects of insulin remains to
be determined. Taken together, the characteristics of APS suggest that
this protein may be an important adapter involved in insulin receptor
signaling in metabolically responsive tissues. The identification of
signaling molecules that are downstream of APS and the physiological
role(s) of APS in insulin-mediated signal transduction is the focus of
further work.