From the Developmental Endocrinology Branch, NICHHD,
National Institutes of Health, Bethesda, Maryland 20892 and
¶ Division of Endocrinology, University of North Carolina Medical
School, Chapel Hill, North Carolina 27514
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ABSTRACT |
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We have previously developed a mouse model of insulin-resistant diabetes by targeted inactivation of the insulin receptor gene. During studies of gene expression in livers of insulin receptor-deficient mice, we identified a novel cDNA, which we have termed sirm (Son of Insulin Receptor Mutant mice). sirm is largely, albeit not exclusively, expressed in insulin-responsive tissues. Insulin is a potent modulator of sirm expression, and sirm mRNA levels correlate with tissue sensitivity to insulin. The product of the sirm gene is a serine/threonine-rich protein with several proline-rich motifs and an NPNY motif, conforming to the consensus sequence recognized by the phosphotyrosine binding domains of insulin receptor substrate and Shc proteins. However, Sirm bears no extended homologies with other known proteins. Based on the sequences of the proline-rich domains, we sought to determine whether Sirm binds to the SH3 domains of FYN and Grb-2. We demonstrate here that Sirm binds to FYN and Grb-2 in 3T3-L1 adipocytes and that insulin treatment results in the dissociation of the Sirm·FYN and Sirm·Grb-2 complexes. We also show that Sirm is a substrate for the kinase activity of FYN in vitro. Based on the patterns of expression of sirm, its regulation by insulin, and the interactions with molecules in the insulin signaling pathway, we surmise that Sirm plays a role in modulating tissue sensitivity to insulin.
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INTRODUCTION |
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We have previously developed a mouse model of insulin-resistant
diabetes by genetic ablation of insulin receptors. Lack of insulin
receptors is rapidly lethal, leading to death from diabetic ketoacidosis within few days of birth (1, 2). We have reported that
insulin treatment of liver extracts derived from insulin receptor-deficient
(ir/
)1
mice resulted in tyrosine phosphorylation of a protein migrating as a
75-kDa species on SDS-PAGE. This protein was not detected in control
(ir+/+ mice) liver extracts. In the course of
experiments designed to determine the identity of this
tyrosine-phosphorylated protein, we isolated a novel cDNA. We
termed the protein product of this cDNA sirm
(Son of Insulin Receptor
Mutant mice). sirm mRNA is most abundant in
insulin-sensitive tissues, such as skeletal muscle, heart, fat, kidney,
and liver, and is regulated by insulin both in vivo and in
cultured cells. In fact, sirm mRNA expression correlates with insulin sensitivity so that it is increased under conditions of
increased insulin sensitivity, for example during differentiation of
3T3-L1 cells and in diabetic ketoacidosis, and is decreased under
conditions of desensitization to insulin, for example following prolonged exposure of cells to insulin. We have cloned a 3661-base pair
sirm cDNA. The amino acid sequence predicted from
sirm cDNA clones encodes a 600-amino acid polypeptide,
bearing no extended homology to other known proteins. The Sirm protein
is rich in serine and threonine residues and contains potential sites
for tyrosine phosphorylation and for phosphorylation by
proline-directed kinases, as well as proline-rich motifs.
Immunoreactive Sirm localizes exclusively to the cytoplasm in skeletal
muscle, whereas it is found partly associated with the membrane
compartment in 3T3-L1 adipocytes. We show, using co-immunoprecipitation
studies and GST binding assays, that Sirm binds to the SH3 domains of
FYN and Grb-2 in vitro and in cultures of 3T3-L1 cells.
Insulin treatment results in the dissociation of the Sirm·Grb-2 and
Sirm·FYN complexes. Sirm also appears to be a substrate for
phosphorylation by the FYN kinase. The engagement of Sirm in complex
formation with adapter proteins and cytoplasmic tyrosine kinases,
together with the distribution of Sirm to insulin-dependent
tissues, and the regulation of sirm mRNA by insulin both
in vivo and in vitro represent circumstantial evidence for the fact that Sirm plays a role in modulating tissue sensitivity to insulin.
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EXPERIMENTAL PROCEDURES |
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Animals-- Husbandry and genotyping of mice bearing a targeted ir allele have been described in previous publications (2-4).
Antibodies-- Anti-phosphotyrosine antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-insulin receptor antibodies were purchased from Oncogene Science (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FYN and anti-Grb-2 antibodies were purchased from Santa Cruz. Anti-Sirm antisera were raised by immunizing rabbits with keyhole limpet hemocyanin-conjugated peptides corresponding to amino acids 136-149 of the Sirm sequence. All antisera were affinity purified using peptide columns. For immunoprecipitation, antibodies were used at the concentrations recommended by the manufacturers. The immune complexes were captured on protein A-agarose beads (Life Technologies, Inc.) and washed three times in 1.0 ml of 50 mM HEPES, 150 mM NaCl, 0.1% Triton X-100 buffer and analyzed by SDS-polyacrylamide electrophoresis. The proteins were transferred to nitrocellulose filters for Western analysis. Blots were pre-blocked in 3% BSA in PBS and incubated with the anti-Sirm antibodies at a 1:1000 dilution in 0.3% BSA in PBS for 2 h at room temperature. Following a short wash in PBS containing 0.05% Tween 20 and 0.05% Triton X-100, a horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad) was added at a 1:5000 dilution and incubated for 20 min. Three additional washes were performed in PBS/Tween/Triton, followed by detection of chemiluminescence using ECL (Amersham Corp.).
RNA Analysis-- Total RNA was isolated from mouse livers and hind limb skeletal muscles or from SV40-transformed hepatocytes using a guanidinium isothiocyanate/acid phenol extraction procedure. Reverse transcription-PCR was performed as described previously. PCR conditions were as follows: denaturation at 94 °C for 1 min, annealing at 45 °C for 1 min, and extension at 72 °C for 1 min for 30 cycles. PCR products were analyzed by gel electrophoresis. For Northern analysis, poly(A)+-enriched RNA was obtained by oligo-(dT) affinity chromatography. 2-3 µg of poly(A)+ RNA were size-fractionated on denaturing 1% agarose, 2.2 M formaldehyde gels and transferred to nitrocellulose membranes. The filters were probed with random-primed sirm cDNA. Additionally, a mouse multiple tissue, an embryonic Northern blot, as well as a zoo blot (CLONTECH, Palo Alto, CA) were used to confirm the identity of the various clones isolated, to study the distribution of the sirm transcripts, and to analyze the presence of sirm gene homologues in other species. Hybridization was performed according to standard procedures.
Cloning of Full-length Sirm cDNA--
Initially, a random
primed sirm fragment obtained by RT-PCR amplification of
ir/
liver RNA was used to probe
106 plaques of mouse embryo and mouse fetal heart cDNA
libraries (CLONTECH). Hybridization and plaque
purification of individual positive clones were carried out according
to standard methods. However, only partial cDNA clones could be
isolated from these libraries. To isolate full-length clones, mouse
heart Marathon-ready cDNA (CLONTECH) was
amplified using primers derived from the 5' and 3' end of the available
sequence. Amplification was carried out as suggested by the
manufacturer, with a mixture of Klenow and Taq polymerase.
Amplified products were subcloned into a PCR II vector (Invitrogen,
Carlsbad, CA) and sequenced using cycle sequencing with fluorescent
dideoxy terminators (Applied Biosystems, Foster City, CA) on 373 and
377 Applied Biosystems sequencers. Each partial clone was hybridized
with multiple tissue Northern blots as well as Northern blots of
ir
/
mice to confirm that amplified sequences
derived from the same original transcripts. Multiple subcloned
fragments were analyzed to confirm the nucleotide sequence. Sequence
alignment was carried out using the Autoassembler software (Applied
Biosystems) to develop a consensus sequence. Ambiguities were further
resolved by visual inspection of electropherograms.
Expression of GST-sirm Fusion Products-- An 1800-nt fragment of the mouse sirm cDNA was amplified from Marathon-ready mouse heart cDNA. The upstream primer 5' GAA TTC TCC AGC TGG CAC ATG AAC AAC AGT3' (nt 949-972) contained an additional EcoRI site in frame with the ATG initiation codon (bold face). The downstream primer spanned nt 2924-2901. The amplified product was digested with EcoRI to generate a 1590-nt fragment, which was subsequently subcloned into the EcoRI site of pGEX2T (Pharmacia Biotech Inc.). The amplified fragment was entirely sequenced to rule out nucleotide substitutions during the amplification procedure. Bacterial cultures were obtained as indicated by the manufacturer, and the recombinant GST-Sirm fusion protein was isolated by affinity chromatography using glutathione-Sepharose beads.
Tissue Isolation-- Whole E13.5 and 18.5 mouse embryos, or livers, hind limb muscles, hearts, epididymal fat pads, and kidneys derived from adult mice were finely minced and homogenized using a glass-glass pestle in 1 mM NaHCO3 containing a mixture of protease inhibitors (Boehringer Mannheim). 20 µg of whole tissue extracts were prepared for SDS-gel electrophoresis as described (2).
Subcellular Fractionation-- In some experiments, subcellular fractions were obtained from cultures of 3T3-L1 cells or from mouse skeletal muscle. After resuspending the cells or tissue in 1 mM NaHCO3 buffer as described above, nuclei were removed by a low speed centrifugation (1000 × g) for 10 min, followed by high speed centrifugation (20,000 × g) to pellet the microsomal fraction and separate the cytosol. The crude microsomal homogenate was solubilized in 50 mM HEPES (pH 7.6) 150 mM NaCl containing 1% Triton X-100 and protease inhibitors, followed by centrifugation at 100,000 × g to remove the insoluble material.
Insulin Stimulation of 3T3-L1 Cells and SV40-transformed
Hepatocytes--
3T3-L1 cells were differentiated as described (5).
Cultures of differentiated 3T3-L1 cells or confluent SV40-transformed hepatocytes derived from ir/
mice or from
ir+/+ mice were grown in 100-mm Petri dishes as
described. Following incubation overnight in 1% dialyzed fetal bovine
serum (3T3-L1) or for 4 h in 1% insulin-free BSA
(SV40-transformed hepatocytes), cells were incubated in the presence or
absence of 100 nM insulin for various lengths of time at
37 °C. Thereafter, cells were frozen in liquid N2 and
thawed in solubilization buffer (1% Triton X-100 in 50 mM
HEPES, 150 mM NaCl, 100 mM NaF, 4 mM sodium orthovanadate, 1 mM EDTA, 4 mM sodium pyrophosphate). For RNA analysis studies, SV40-transformed hepatocytes were incubated with insulin (1 or 100 nM) for 4 h, frozen in liquid N2, and
thawed for RNA extraction as outlined above.
GST Fusion Protein Binding Assay-- The GST fusion proteins used in these studies are as follows: mouse c-Abl type IV SH3 domain (amino acids 84-138) (6), the mouse phosphatidylinositol 3-kinase p85 SH3 domain (amino acids 1-80) (7), the human FYN SH3 domain (amino acids 84-148) (8), and the full-length mouse Grb2 (Upstate Biotechnology). Confluent monolayers of SV40-transformed hepatocytes (in two 175-cm2 flasks) were solubilized in 5 ml of 1% Triton buffer. 5 µg of recombinant GST fusion protein in 0.05 ml were added to the extracts and allowed to bind for 1 h at 4 °C on a rotating wheel. Thereafter, the material was pelleted by centrifugation and washed in 10 ml of 0.1% Triton buffer four times. The final pellet was prepared for electrophoresis as described above.
In Vitro Kinase Activity--
2 units of purified FYN kinase
(Upstate Biotechnology) were incubated with 1 unit of denatured rabbit
muscle enolase (Sigma) in the presence of GST, or GST-Sirm, or with
various concentrations of peptides corresponding to Sirm amino acid
sequences 224-246 (peptide 1), 331-339 (peptide 2), and 29-40
(peptide 3). Thereafter, a phosphorylation buffer containing 50 mM HEPES (pH 7.4), 5 mM MgCl, and 10 µCi of
[-32P]ATP (3000 Ci/mmol, NEN Life Science Products)
was added, and the reaction was incubated at room temperature for 30 min. The reaction was stopped by adding Laemmli sample buffer, and the products were analyzed by SDS-PAGE followed by autoradiography. One of
two experiments is shown.
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RESULTS |
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Cloning of Sirm--
We had previously shown that insulin
stimulation of liver extracts derived from
ir/
mice results in tyrosine phosphorylation
of a protein of apparent mass of ~75 kDa that is not observed in
ir+/+ mice (see Fig. 3B in Ref. 2).
We questioned whether this protein may share sequence similarity with
the insulin receptor or its substrates. To this end, we carried out
RT-PCR amplification from ir
/
and
ir+/+ mice liver RNA using several sets of
primers, the sequences of which were derived from mouse insulin and
IGF-1 receptors, as well as insulin receptor substrate-1 (IRS-1). A set
of primers designed to amplify the extracellular domain of the IGF-1
receptor consistently gave rise to an additional band of slightly
larger size than predicted (Fig.
1A). This band was unique to
ir
/
liver RNA and was never observed in
amplification experiments with ir+/+ mouse liver
RNA (data not shown). The band was isolated, sequenced, and used as a
hybridization probe to isolate clones containing the sirm
coding sequence. By using a combination of cDNA library screening
and cDNA amplification techniques, we cloned a 3661-nt cDNA,
with an open reading frame of 1800 nucleotides, predicted to encode a
600-amino acid polypeptide. The 5' end of the cDNA was isolated
from mouse heart Marathon cDNA (CLONTECH). Two
different clones were isolated, possibly as the result of two different transcription start sites. The longest cDNA contains 962 nt of 5'-untranslated sequence; the shorter one contains 620 nt. Two non-canonical ATTAAA polyadenylation signals are found 24 and 160 nt
downstream of a putative ochre termination codon. In vitro translation of a full-length sirm cDNA clone in a rabbit
reticulocyte lysate yielded a peptide of 81 kDa (Fig.
1B).
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sirm mRNA Is Overexpressed in Liver and Skeletal Muscle of Mice
Lacking Insulin Receptors--
sirm was originally detected
in RNA derived from ir/
mice. Next, we asked
whether failure to detect sirm in the same assay in
ir+/+ mice could be due to overexpression of
sirm in ir
/
mice compared with
normal littermates. Northern blotting analysis was performed on pooled
RNAs derived from skeletal muscle and liver of three to five
ir+/+, ir+/
, and
ir
/
mice (Fig.
3). In liver of
ir
/
mice, sirm mRNA levels
were 3-fold higher than in ir+/+ mice (Fig. 3,
lanes 4 and 6) after correcting for the amount of
actin mRNA (lower panel), whereas in skeletal muscle
they were 40% higher (lanes 1 and 3).
Intermediate variations were observed in ir+/
mice (lanes 2 and 4).
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Expression of Sirm Correlates with Tissue Sensitivity to
Insulin--
To determine further whether expression of
sirm is under regulation by insulin, we studied the effect
of insulin on sirm expression in SV40-transformed
hepatocytes derived from ir/
and
ir+/+ mice. Cells derived from
ir
/
mice are devoid of insulin receptors but
possess about 50,000 IGF-1
receptors/cell.2 After
treatment with insulin (1 or 100 nM), RNA was isolated and
analyzed by Northern blotting with a 32P-labeled
sirm cDNA (Fig. 4). 4 h treatment of normal cells with 1 nM insulin led to a 50%
decrease of sirm expression, and 100 nM insulin
led to a 90% decrease (Fig. 4, lanes 1-3). In contrast, in
ir
/
cells, 1 nM insulin
decreased sirm mRNA levels by 20% and 100 nM insulin by 45% (Fig. 4, lanes 4-6). Thus,
expression of the sirm gene is decreased following prolonged
exposure of cells to insulin. The ID50 for insulin
inhibition of sirm expression is ~1 nM in
ir+/+ cells and ~100 nM in
ir
/
cells. This observation suggests that
insulin regulates sirm expression in
ir
/
cells by binding to IGF-1 receptors. In
fact, insulin would be predicted to bind to IGF-1 receptors with about
100-fold lower affinity compared with insulin receptors.
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Sirm Is Prevalently Expressed in Insulin-responsive Tissues-- We performed a tissue survey of sirm gene expression (Fig. 5). During mouse embryonic development, sirm expression peaks in mid-late gestation (E11-15), to then decline prior to birth (left panel). In adult mouse, sirm is most abundant in skeletal muscle and heart (Fig. 5, middle panel). Additionally, sirm is expressed in kidney, brain, and lung. Although not clearly visible on this exposure of the blot, sirm mRNA can be detected in liver, as demonstrated in Figs. 3 and 4. Expression of sirm is associated with adipocyte differentiation of 3T3-L1 cells (Fig. 5, right panel). Furthermore, sirm is identical to EST T10445, which was isolated from a pancreatic islet library (11, 19). Thus, sirm is expressed in virtually all insulin-responsive tissues and, at lower levels, in other organs. Similar expression patterns were detected using Northern analysis of human RNAs (data not shown). A zoo blot (CLONTECH) was hybridized to a 32P-labeled mouse sirm cDNA. Results indicate that homologues of the mouse sirm gene are present in human, rat, and yeast (data not shown).
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Subcellular Distribution of Sirm and Quantification of the Protein Product in Mice-- We raised a polyclonal antserum against the peptide sequence corresponding to amino acids 136-149 of Sirm. The antiserum detected an immunoreactive species of ~90 kDa in several mouse tissues, which is specifically competed by the peptide used for affinity purification of the antiserum (Fig. 6A, compare left and middle panel). The same band is not present when preimmune serum is used for immunoblotting (Fig. 6A, right panel). The tissue distribution of immunoreactive Sirm protein correlates well with patterns of mRNA expression.
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Sirm Binds to Grb-2 and to the SH3 Domain of FYN in Vitro--
The
amino acid sequence of Sirm contains four proline-rich domains that
could potentially serve as binding sites for SH3-containing proteins.
Based on the homology between these domains and the known binding
specificities of Grb-2 and FYN (Fig. 2B), we investigated whether Sirm could bind to Grb-2 or FYN SH3-GST fusion proteins in vitro. Furthermore, in view of the consensus sequence
among Sirm (amino acids 224-246) and several Abl-binding proteins, we also included the Abl SH3-GST protein in these experiments, as well as
the p85 SH3-GST as a negative control. GST fusion proteins containing the SH3 domains of the p85
subunit of
phosphatidylinositol 3-kinase (7), Abl (6), FYN (8), and full-length
Grb-2 were expressed in Escherichia coli, purified, and
incubated with detergent extracts of SV40-transformed hepatocytes.
Immunoblotting of the resulting gels with the Sirm antibody showed that
Sirm binds to GST-Grb2 and GST-FYN SH3 but not to the SH3 domains of p85
or Abl (Fig. 7, left
panel). To characterize better the nature of this interaction, we
incubated in vitro translated Sirm with GST fusion proteins
in the absence or presence of two proline-rich peptides derived from
the Sirm sequence (Fig. 7, right panel). Sirm binding to
Grb-2 was inhibited by incubating extracts in the presence of the
proline-rich peptide IPLLKSPLLPLPTPKS (peptide 1, Fig. 7)
but not by peptide NPLPTTPKR (peptide 2 Fig. 7), whereas binding to FYN SH3 was inhibited by both peptides, indicating that, at
least in this reconstitution experiment, Sirm can interact with Grb-2
and FYN directly, through one (Grb-2) or more (FYN) proline-rich
sequences (Fig. 2).
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Sirm Binds to Grb-2 and FYN in 3T3-L1 Cells-- Insulin treatment dissociates the sirm·Grb-2 and sirm·FYN complexes. Next, Sirm binding to SH3 domain proteins was investigated in cultures of 3T3-L1 cells. We questioned whether the association between Sirm and Grb-2 or FYN could be detected in vivo by way of co-immunoprecipitation assays (Fig. 8). As shown in A, Sirm expression is greatly increased following differentiation of 3T3-L1 cells and is decreased by ~30% by treatment with 100 nM insulin for 30 min. Following immunoprecipitations with the various antibodies, proteins bound to the immune complexes were analyzed by SDS-PAGE and immunoblotting with anti-Sirm antibody. Following immunoprecipitation with FYN antibody (Fig. 8B), immunoreactive Sirm could be detected in differentiated 3T3-L1 cells under basal conditions. However, insulin treatment resulted in the complete disappearance of FYN-bound Sirm, indicating that FYN is indeed complexed with Sirm in 3T3-L1 cells and that insulin treatment causes dissociation of the Sirm·FYN complexes. Likewise, Sirm could be detected in Grb-2 immunoprecipitates under basal conditions but not following insulin treatment (Fig. 8C). No Sirm immunoreactivity is present following immunoprecipitation with nonimmune serum (Fig. 8D). Control immunoblotting experiments with Grb-2 and FYN antibodies indicate that the same amount of protein is present in all lanes (not shown).
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Sirm Is a Substrate for the Kinase Activity of FYN in Vitro, the
Failure of Sirm to Modulate FYN Kinase--
The activity of Src family
kinases, such as FYN, is regulated through interactions of cellular
proteins with their SH2 and SH3 domains. For example, deletion of
either domain leads to constitutive activation of the Abl or Src
kinases (20-22). We wanted to determine whether Sirm could modulate
FYN kinase activity through SH3 domain interactions. GST-Sirm (amino
acids 1-535) was incubated with partially purified FYN and
[-32P]ATP (Fig. 9), and
the kinase activity of FYN was measured using enolase as a substrate in
the presence or absence of various concentrations of the two
proline-rich peptides that interact with FYN (Fig. 9, lanes
4-9). Neither GST-Sirm (amino acids 1-535) nor Sirm-derived peptides altered phosphorylation of enolase by FYN. Furthermore, FYN
catalyzed the phosphorylation of GST-Sirm (lane 3). We have also shown that Sirm can serve as a substrate for the insulin and IGF-1
receptor tyrosine kinases in vitro (not shown).
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DISCUSSION |
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In this paper, we report the identification and a preliminary characterization of a novel gene, which we have termed sirm. Even though the identification of sirm was the serendipitous by-product of a search to identify proteins uniquely expressed by insulin receptor-deficient mice, several features of the sirm gene prompted us to investigate it further. First, insulin is a potent regulator of sirm mRNA expression, so that sirm mRNA is up-regulated in conditions of increased tissue sensitivity to insulin, such as differentiation of 3T3-L1 cells and diabetic ketoacidosis, whereas it is down-regulated by prolonged insulin treatment. Interestingly, the highest levels of sirm expression are found in insulin-sensitive tissues, such as skeletal muscle, heart, fat, kidney, and liver. Indirect evidence indicates that sirm is also expressed in pancreatic beta cells, since an EST with virtual sequence identity to sirm was detected in a beta cell library (11). Interestingly, we have localized the human homologue of sirm to chromosome 15q15, in the genetic interval between D15S118 and D15S123, which corresponds to 32-45 centimorgans on the physical map of chromosome 15.3 This region contains marker D15S102. In studies of Mexican American sibling pairs with non-insulin-dependent diabetes mellitus, Hanis et al. (23) reported a significant departure from the expected frequency of allele sharing at this marker. Further studies will be necessary to determine whether this region contains a diabetes-susceptibility locus; however, the co-localization of SIRM to this region is intriguing.
The sequence of Sirm contains a number of interesting features that suggest clues as to its function. In this paper, we have shown that Sirm can interact with proteins containing SH3 domains through its proline-rich motifs. In 3T3-L1 cells, Sirm associates with the Src family tyrosine kinase FYN. Binding of Sirm to FYN was predicted on the basis of a consensus binding site for the FYN SH3 domain (Fig. 2B) (14, 24). Interestingly, insulin treatment leads to complete dissociation of Sirm from FYN. The binding of Sirm to FYN is intriguing, in view of the fact that FYN has been implicated in adipocyte-specific functions of insulin and particularly in the mechanism whereby insulin stimulates phosphorylation of caveolin in 3T3-L1 cells (25, 26). Caveolin is an important component of caveolae, flask-shaped invaginations of plasma membranes, which are thought to participate in the processing of intracellular signals. Some workers (27) have also reported that caveolae contain adapter molecules like Grb-2, which also binds to Sirm. It remains to be determined whether Sirm co-localizes with FYN in caveolae; however, it is interesting that a significant fraction of cellular Sirm is associated with the plasma membrane in 3T3-L1 adipocytes. Work from the Saltiel laboratory (26) has suggested that Cbl is the kinase responsible for stimulating the adipocyte-specific caveolin kinase activity of FYN. FYN kinase is activated by binding of cellular proteins to its SH2 and SH3 domains. Thus, an appealing possibility was that Sirm would regulate the FYN kinase in an adipocyte-specific fashion. We have failed to demonstrate that Sirm or peptides derived from it can modulate the kinase activity of FYN toward enolase. While these findings do not entirely rule out that Sirm may modulate the FYN kinase in vivo, they are consistent with data of Mastick et al. (26), suggesting that the kinase activity of FYN is not different in differentiated 3T3-L1 cells compared with their undifferentiated counterpart. On the other hand, the identification of Sirm as an adipocyte-specific substrate of FYN raises the interesting possibility that Sirm may participate in adipocyte-specific functions of FYN.
The binding of Sirm to Grb-2 is also consistent with the known specificity of the Grb-2 SH3 binding domain (Fig. 2B) (28). The dissociation of Sirm from Grb-2 follows the same time course and dose responsiveness as insulin-induced dissociation of Grb-2 from son-of-sevenless (29, 30). This dissociation has been invoked as a possible mechanism underlying the transient nature of insulin's effect to activate Ras. By analogy, one could envision that Sirm links Grb-2 to downstream effectors and that this effect is lost upon prolonged insulin stimulation.
The evidence presented in this paper indicates that Sirm is a novel adapter protein, which can function in a variety of cell types by binding the Src family kinase FYN and Grb-2. Sirm expression appears to correlate with the ability of organs and cells to respond to insulin, and several of the intracellular targets of Sirm have been implicated in signaling downstream of the insulin receptor. We would like to tentatively propose that Sirm plays a role in modulating insulin action in insulin-sensitive tissues via its association with signaling molecules. Further studies required to conclusively test this hypothesis are under way.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. I. Taylor for support during the initial phase of these studies. We also thank M. Cool for expert technical assistance and Dr. P. Goldsmith for help with the purification of the antisera. The GST-FYN, p85, and FYN-SH3 fusion proteins were obtained from Drs. B. Mayer, H. Band, and L. Cantley through the courtesy of Dr. S. Shoelson. We are indebted to Dr. S. M. Najjar for critical reading of the manuscript.
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FOOTNOTES |
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* These studies were supported in part through a research grant of the American Diabetes Association and a generous gift of Sigma Tau Corp. (to D. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U59739.
§ Current address: Facoltá di Scienze Ambientali, Seconda Universitá di Napoli, Caserta, Italy.
To whom correspondence should be addressed: Bldg. 10, Rm.
10D18, Bethesda, MD 20892. Tel.: 301-496-9595; Fax: 301-402-0574; E-mail: accilid{at}cc1.nichd.nih.gov.
1 The abbreviations used are: ir, insulin receptor; Grb-2, growth factor receptor binding protein-2; BSA, bovine serum albumin; SH3, src homology 3 domain; IGF-1, insulin-like growth factor type 1; IRS, insulin receptor substrate; GST, glutathione S-transferase; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; EST, expressed sequence tags; nt, nucleotide(s).
2 K. Rother, Y. Imai, and D. Accili, manuscript in preparation.
3 Y. Kido, and D. Accili, manuscript in preparation.
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REFERENCES |
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