The Insulin Receptor Substrate (IRS)-1 Pleckstrin Homology Domain Functions in Downstream Signaling*

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

After insulin binds to its receptor, it activates an intrinsic tyrosine kinase activity, which mediates the tyrosine phosphorylation of a variety of endogenous substrates including insulin receptor substrates (IRS) 1 1-4 (1, 2). Binding of these tyrosine phosphorylated substrates to the Src homology (SH) domain-2 of the regulatory subunit of the heterodimeric p85/p110 phosphatidylinositol (PI) 3-kinase leads to a 3-5-fold stimulation in its enzymatic activity and an increase in the PI 3,4-bisphosphate and 3,4,5-trisphosphate in the cell (3,4). As a result of the increase in these lipids, there is a pronounced stimulation in the enzymatic activity of the Ser/Thr kinases called Akt, and a more modest increase in the enzymatic activity of particular isoforms of protein kinase C, which ultimately lead to numerous biological responses (5,6).
The IRS 1-4 all contain an amino-terminal pleckstrin ho-mology (PH) domain next to a phosphotyrosine binding (PTB) domain (1). The PH domain is homologous to a region in pleckstrin and has been found in over 120 proteins including serine/ threonine kinases, tyrosine kinases, phospholipases, GTPaseactivating proteins, GTPases, and cytoskeletal proteins (7). PH domains are often involved in the attachment of proteins to membranes, either by directly binding to phospholipids and/or by protein-protein interactions with, for example, the ␤␥ subunits of the heterotrimeric G proteins (8). In the case of the PH domain of IRS-1, both phospholipid binding as well as protein binding have been reported (9 -11).
In contrast, the PTB domain of IRS-1 has been documented to bind to phosphotyrosines in the motif found in the juxtamembrane region of the insulin receptor (i.e. NPXpY) (12,13). Although the binding specificities of these two regions is quite distinct, they share a similar overall structure, with each containing a ␤-sandwich formed by two nearly orthogonal antiparallel ␤-sheets of 4 and 3 strands, respectively. In recent studies, the crystal structure of the region of IRS-1 containing both the PH and PTB domains has been determined (14). These data indicate that the two binding domains may act cooperatively to localize IRS-1 at the membrane in association with the receptor and thereby allow multiple tyrosine phosphorylations of IRS-1.
A variety of experimental approaches have also been utilized to test the role of the PH and PTB domains of the IRS proteins in their interactions with the IR and the subsequent tyrosine phosphorylations. Mutant IRS molecules in which either the PH or PTB domain have been deleted or replaced with homologous structures of other proteins have been expressed in mammalian cells (15)(16)(17)(18). These studies have documented a primary role for the PH domain in the subsequent ability of IRS-1 to be tyrosine phosphorylated in intact cells after insulin stimulation. In either in vitro studies or yeast two-hybrid systems, a primary role of the IRS-1 PTB domain has been documented in receptor interactions (15,19). In the case of IRS-2, a more central region of the molecule has also been identified as playing a role in receptor interactions (20,21).
Although numerous studies have documented the roles of the PH and PTB domains of the IRS proteins in the interactions with the IR and the subsequent tyrosine phosphorylation of the IRS proteins, no studies have tested the roles of these two domains on subsequent signals induced by the IRS⅐PI 3-kinase complex. Because mutations in the PH and PTB domains interfere with the interaction of the IRS with the IR and its subsequent tyrosine phosphorylation, it is impossible to determine the effect of these mutations on the downstream signals emanating from the IRS⅐PI 3-kinase complex. To overcome this obstacle, we now describe a constitutively active form of IRS-1. In this chimeric molecule, the inter-SH2 domain of the p85 regulatory subunit of PI 3-kinase is attached to various portions of the IRS-1 molecule. The inter-SH2 domain of p85 has previously been documented to bind to the 110-kDa catalytic unit of the PI 3-kinase (22). Thus the resultant chimeric IRS-1 molecules now constitutively bind to the PI 3-kinase. By using these chimeric molecules, we have tested the role of different regions of the IRS-1 molecule in eliciting a subsequent biological response, the stimulation of the Ser/Thr kinase Akt. We demonstrate that the PH domain alone is sufficient to target the constitutively active PI 3-kinase to induce the activation of Akt and that this requires the lipid binding properties of the PH domain.

EXPERIMENTAL PROCEDURES
Reagents-Cell culture medium DME-H21 and oligonucleotides were from Life Technologies, Inc. Glutathione-agarose beads and monoclonal anti-GST antibodies were from Sigma, protein A-Sepharose was from Repligen (Cambridge, MA), 12CA5 monoclonal anti-HA antibodies were from Roche Molecular Biochemicals (Mannheim, Germany), rabbit polyclonal anti-Akt antibodies directed against the PH domain of Akt1 were produced as described (23 Cell Culture and Treatments-3T3-L1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum at 37°C, 5% CO 2 . The cells were grown to confluency and serum-starved 2-4 h before each experiment. Insulin was added when needed as described in the figure legends.
DNA Constructs-DNA fragments encoding the iSH2 domain of the p85 subunit of PI 3-kinase with the HA tag were generated by PCR and inserted into the retroviral vector pWZL-hygro using EcoRI and SalI sites. The obtained intermediate pWZL-iSH2-HA plasmid was used for insertion of the various domains of IRS-1 (PH, PH-PTB, PTB). These amino-terminal parts of the constructs were generated by PCR using a set of 5Ј and 3Ј primers containing BamHI and EcoRI recognition sites attached to the downstream sequence of interest. Resultant PCR products were then ligated into pWZL-iSH2-HA. The iSH2 construct was obtained in a separate PCR reaction by introducing an ATG codon in front of the iSH2 sequence using pWZL-iSH2-HA as a template. The R28C single amino acid substitution was obtained by mutagenesis using the QuickChange mutagenesis kit (Stratagene, Inc.) and the PH-PTB⅐iSH2 cDNA as a template. Two additional amino acids changes (K21L and K23L were added in a single round of mutagenesis using the same kit and the R28C mutant cDNA as a template to generate the triple mutant (TM). Restriction endonuclease mapping was used for screening of colonies because an EcoR47III site was destroyed during the mutagenesis. DNA sequence analyses were performed to verify the sequences of the constructs. To generate the GST fusions of PH, PH-PTB, and single and triple mutant PH-PTB, the appropriate portions of the IRS-1 constructs of the retroviral plasmids were cut out and inserted into the pGEX-4T-3 vector.
Protein Expression in Escherichia coli and Purification of GST Fusion Proteins-E. coli BL-21 cells transfected with pGEX vectors encoding the IRS-1 constructs were grown to mid-log phase, induced with 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside and grown for an additional 3-4 h. Cells from one liter of bacterial cultures were harvested by centrifugation at 7700 ϫ g for 10 min at 4°C (Beckman JA-10 rotor), resuspended in 20 ml of phosphate-buffered saline buffer containing protease inhibitors and sonicated on ice in short bursts. Sonicated bacterial extracts were solubilized for 30 min at 4°C using 1% Triton X-100 and centrifuged at 12,000 ϫ g for 10 min. Cleared supernatants were incubated with glutathione-agarose beads (Sigma) for 2 h at 4°C. Beads with immobilized complexes were washed several times with phosphate-buffered saline and then eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0 for 30 min at 4°C. Purified fusion proteins were dialyzed against phosphate-buffered saline and analyzed by 15% SDS-polyacrylamide gel electrophoresis. The purity of the proteins was assessed by densitometry of the Coomassie-stained gels.
Generation of Stable Cell Lines Expressing the IRS-1⅐iSH2 Constructs-3T3-L1 fibroblasts were infected with the retroviruses express-ing IRS-1⅐iSH2 constructs as previously described (26). In brief, 70% confluent Phoenix packaging cells were transiently transfected with plasmid DNA (pWZL-constructs) using the calcium phosphate precipitation method as described (26). After the final medium change, the cells were incubated for 3 days at 30°C, 5% CO 2 to generate the viral supernatant. Viral infection of 3T3-L1 cells was performed using supernatants collected from the transfected Phoenix cells. 6-well plates with 50% confluent 3T3-L1 cells were infected with 1 ml of each viral supernatant. The plates were spun at 2,500 rpm for 90 min and then cultured until confluent. Cells were selected in complete medium containing 500 g/ml hygromycin for a total of 8 days. The total pool of selected cells was used in all subsequent experiments.
Western Blot Analyses-3T3-L1 fibroblasts overexpressing IRS-1⅐iSH2 constructs were grown in 10-cm plates to confluence and lysed in Akt lysis buffer (see Akt kinase assay below). After removal of the insoluble matter by centrifugation, the cell lysates were immunoprecipitated using anti-HA epitope antibodies (12CA5) bound to protein A-Sepharose beads. Precipitated complexes were washed three times with cold buffer containing 50 mM Hepes, pH 7.6, 150 mM NaCl, and 0.1% Triton X-100. Twenty microliters of gel loading buffer were added to each immunoprecipitate, and the samples were boiled for 5 min and loaded on 10% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose membranes and blotted with 12CA5 antibodies, and bound mouse antibodies were visualized using an anti-mouse antibody conjugated to horseradish peroxidase and ECL. 3T3-L1 cells stably transfected with the empty pWZL vector were used as a negative control.
Subcellular Fractionation-3T3-L1 cells expressing the PH-PTB⅐iSH2 constructs were grown to confluence and serum-deprived for 2 h. The cells were washed rapidly with ice-cold TES (20 mM Tris, pH 7.4, 5 mM EDTA, 250 mM sucrose), and then harvested in 1 ml of homogenization buffer containing TES with 10 g/ml aprotinin, 10 g/ml leupeptin, 250 M phenylmethylsulfonyl fluoride, 2 mM sodium vanadate, 10 mM sodium fluoride, and 2 mM sodium pyrophosphate. The cell suspension was homogenized by passaging 10 times through a 25-gauge needle, incubated on ice for 10 min, and then spun at 4°C for 10 min at 3000 rpm on a table top centrifuge. Supernatants were removed and subjected to ultracentrifugation at 100,000 ϫ g (60,000 rpm) for 1 h at 4°C using a TLA-100.3 rotor (Beckman Instruments Inc.). Supernatants obtained in the high-speed spin, designated cytosol (C), were immunoprecipitated with anti-HA antibodies. Pellets were resuspended in 1 ml of the homogenization buffer and spun at 100,000 ϫ g for 1 h. Supernatants obtained in the second high-speed spin were discarded, and pellets were solubilized on ice in homogenization buffer containing 0.5 M NaCl for 30 min. Solubilized pellets were then recentrifuged at 14,000 rpm for 15 min at 4°C. Supernatants obtained in this spin, designated as soluble particulate (SP), were also immunoprecipitated. The pellets from this spin (designated as insoluble particulate, IP) were dissolved by boiling in SDS and directly loaded onto 10% SDS-polyacrylamide gels. Immunoprecipitates of cytosol (C) and soluble particulate (SP) were loaded on the same gel, and all three subcellular fractions were analyzed by immunoblotting with 12CA5 antibodies and ECL. Protein bands were quantified using an NIH image program. Ratios of the total particulate fraction over cytosol were calculated for each construct.
Akt and PI 3-Kinase Assays-3T3-L1 cells expressing IRS-1⅐iSH2 constructs were grown to confluence, serum-deprived for 2 h and stimulated with insulin as indicated. Cells were then washed once with cold HBS buffer (50 mM Hepes, pH 7.6, 150 mM NaCl) and lysed in either PI 3-kinase lysis buffer (137 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM MgCl 2 , 1 mM CaCl 2 , 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 400 M vanadate, 1 mM dithiothreitol) or Akt lysis buffer (50 mM Hepes, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM vanadate, 2 mM sodium pyrophosphate, 30 mM sodium fluoride, 1 mM EDTA, 1 mM dithiothreitol, 100 nM okadaic acid). For the PI 3-kinase assays, cell lysates were immunoprecipitated with the anti-HA epitope antibodies (12CA5) captured on protein A-Sepharose beads. The precipitates were assayed for PI 3-kinase activity as described (27) using phosphatidylinositol as a substrate. The amounts of PI 3-kinase activity in the cells transfected with pWZL vector were considered as background (always less than 5% of the signal) and subtracted from experimental values. Data shown are the means of three independent experiments. In the Akt kinase assays, endogenous Akt was immunoprecipitated from cell lysates using an antibody to the PH domain of Akt bound to protein A-Sepharose. Akt assays were performed as described in Ref. 28  Forty microliters of the reconstituted phospholipids were incubated with 0.5 g of each protein in 100 l of HNE buffer containing 0.02% Nonidet P-40. The mixtures were incubated for 1 h at room temperature. Then 35 l of the glutathione-agarose slurry were added to each reaction and incubated for 30 min at 4°C to capture the proteinphospholipid complexes. Beads were washed once with 1 ml of the cold HNE buffer containing 0.5% Nonidet P-40, and radioactivity retained on the beads was measured by liquid scintillation counting (Beckman LS).

Construction and Expression of Chimeric IRS-1
Molecules-To generate IRS-1 molecules that would constitutively bind and activate PI 3-kinase without being tyrosine phosphorylated, we constructed cDNAs that encode different portions of the IRS-1 molecule fused in frame with the inter-SH2 domain of the p85 regulatory subunit of the PI 3-kinase because this peptide (22) has previously been documented to be sufficient to bind to the 110-kDa catalytic unit of the PI 3-kinase (Fig. 1). In addition, each chimera contained an HA tag at the carboxyl terminus. Stable cell lines expressing each of these constructs were generated, and the amount of the expressed construct was examined by immunoprecipitation and Western blotting (Fig.  2). The construct only encoding the inter-SH2 domain was found to be poorly expressed and not studied further (Fig. 2). In contrast, constructs encoding either the PH domain alone or both the PH and PTB domains were found to be well expressed although the construct encoding the PH domain alone was expressed at a level of about one-half that of the PH-PTB construct (Fig. 2). In addition, point mutants in the PH domain of the PH-PTB construct were made. These included both a single mutant in which Arg 28 was changed to Cys (called PH-(R28C)-PTB) or a triple mutant containing the Arg 28 mutation as well as mutations in Lys 21 and Lys 23 (called PH-(TM)-PTB). Both of these mutants were found to be expressed at levels comparable with the nonmutant PH-PTB construct (Fig. 2). These mutations were designed based on the findings that mutations in comparable residues of other PH domains impairs their ability to function (7). Finally, a construct that only con-tained the PTB domain fused to the inter-SH2 domain was found to be expressed although at a lower level then the constructs containing either both the PH and PTB domains or only the PH domain (Fig. 2).
Measurement of the Amount of PI 3-Kinase Activity Associated with the Different Chimeric IRS-1 Molecules-To determine whether the chimeric IRS-1 molecules did contain an associated PI 3-kinase activity, lysates of the stable cell lines described above were immunoprecipitated with an antibody to the HA epitope, and the amount of PI 3-kinase activity in these precipitates was determined (27). The construct containing both the PH and PTB domains of IRS-1 had the maximal amount of PI 3-kinase activity associated with it (Fig. 3). The amount of PI 3-kinase activity associated with the point mutants in the PH domain of the PH-PTB construct (both PH-(R28C)-PTB and PH-(TM)-PTB) were found to be not significantly different then the nonmutant PH-PTB domain containing chimera (Fig. 3). The chimeric construct containing only the PH domain of the IRS-1 had ϳ50% of the PI 3-kinase activity of the PH-PTB molecule, whereas the PTB and iSH2 constructs had lower amounts of PI 3-kinase activity. The PI 3-kinase activities associated with these constructs (Fig. 3) were proportional to the amounts of each protein expressed (Fig. 2).
Measurement of the Amount of Active Akt Induced by the Chimeric IRS-1 Molecules-To test the role of the different domains of IRS-1 in the ability of the chimeric molecule to stimulate a downstream response, we examined the amount of active endogenous Akt in the cell lines expressing the different constructs. Confluent cells were serum-deprived for 2 h, and then cells expressing the pWZL vector were stimulated with insulin to determine the maximal levels of active endogenous Akt in these cells. Cells were lysed, and the Akt was immunoprecipitated and assayed for enzymatic activity (28). Cells expressing the PH-PTB domain had the highest levels of endogenous Akt activity, a value ϳ60% of that observed after stimulation with insulin (Fig. 4). Mutation of either a single residue in the PH domain (the PH-(R28C)-PTB construct) or 3 amino acids (the PH-(TM)-PTB construct) in the PH-PTB construct significantly impaired the ability of these constructs to activate the endogenous Akt (Fig. 4). In cells expressing a construct that only contained the PH domain, the endogenous Akt was also activated to a level of ϳ40% of that observed after insulin stimulation (Fig. 4). The construct containing only the PTB domain of IRS-1 exhibited a low ability to activate the endogenous Akt, consistent with its low expression and poor activation of the PI 3-kinase.
Testing the Effect of the Mutations in the PH Domains on its Lipid Binding Activities-To test the lipid binding activities of the various mutant PH domains, we produced bacterial GST fusion proteins with the wild-type PH-PTB molecule (lacking the inter-SH2 domain of the p85 subunit of PI 3-kinase) as well as the single point mutant (R28C) and the triple mutant (R28C/ K21L/K23L). The purified GST fusion proteins were incubated with labeled PI 4,5-P 2 , washed, and counted. The PH-PTB molecule bound substantially more lipid then the control GST protein (Fig. 5). In addition, the two mutant GST⅐PH-PTB proteins bound ϳ75% less lipid than the wild-type PH-PTB protein.
Testing the Effect of the Mutations in the PH Domains on the Subcellular Distribution of the Chimeric IRS-1 Molecules-To test the effect of the point mutations in the PH domains on the subcellular localization of the chimeric IRS-1 molecules, we fractionated cells expressing either wild-type PH-PTB construct, PH-(R28C)-PTB or PH-(TM)-PTB. The fractions were tested for the presence of the constructs by Western blotting. We found that the wild-type PH-PTB IRS-1 chimera was present in the particulate fraction to a much greater degree then either the single mutant (PH-(R28C)-PTB) or the triple mutant (PH-(TM)-PTB); the particulate to cytosolic ratio of the wildtype PH-PTB chimera was 2.5-fold greater then the comparable ratio for either PH-(R28C)-PTB or PH-(TM)-PTB (Fig. 6). DISCUSSION The principal mechanism whereby the insulin receptor activates PI 3-kinase appears to be via its ability to stimulate the tyrosine phosphorylation of various endogenous proteins including the different IRS molecules, IRS-1 to 4 (1, 2). Activation of PI 3-kinase plays a critical role in subsequent biological responses, such as stimulation of glucose uptake and regulation of gene transcription (5,6). A number of downstream targets of the PI 3-kinase and the lipids it generates have been identified. These include several Ser/Thr kinases such as the PDK-1, the family of Akt/PKB kinases, and several atypical protein kinase C including PKC and . The exact role of these different kinases in eliciting subsequent biological responses has been debated (5, 6). Extensive studies have investigated the mechanism whereby the IRS proteins are localized to the insulin receptor. All four IRS molecules contain both a PH domain and a PTB domain (1,2). The PH domain binds various phospholipids, including PI 4,5-P 2 as well PI 3,4,5-P 3 (9,10). In addition some studies have suggested that the PH domain may also interact with various proteins (11). In contrast, the PTB domains bind phosphotyrosines, in particular, a phosphotyrosine in the juxtamembrane region of the insulin receptor (12,13). Based on the crystal structure of the PH and PTB domains of the IRS-1 molecule, a model has been proposed of the two domains binding cooperatively to localize IRS-1 at the membrane in association with the insulin receptor (14).
In the present studies, we have tested the role of these two domains in the ability of the IRS-1 molecule to elicit a subsequent biological response, the activation of the Ser/Thr kinase Akt. To accomplish this, we produced novel chimeric molecules, which contain these two domains fused to the inter-SH2 domain of the p85 subunit of the PI 3-kinase. This peptide has previously been shown to be sufficient to bind and activate the PI 3-kinase (22). By adding these residues to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules, which were constitutively bound to active PI 3-kinase. This was necessary because prior studies have shown that the PH domain of the IRS-1 molecule was critical for its subsequent tyrosine phosphorylation and association with the PI 3-kinase. By adding the inter-SH2 domain to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules that were active in the absence of insulin stimulation and receptor-mediated tyrosine phosphorylation.
In the present work we have shown that the PH domain is critical for the subsequent ability of these constitutively active chimeric molecules to activate Akt. The most convincing data for this conclusion comes from the studies of the point mutants of the PH-PTB domain chimeric molecule. The wild type and mutants were expressed to comparable levels and had almost identical associated PI 3-kinase activities. However, the wild type activated the endogenous Akt whereas both the single and triple PH domain mutants had a dramatically decreased ability to activate the enzymatic activities of the endogenous Akt (i.e. the introduction of either a single mutation or 3 mutations in the PH domain caused an approximate 75% decrease in the ability of the PH-PTB construct to activate Akt). Both mutations also had a similar decreased ability to bind to PI 4,5-P 2 in comparison to the wild-type protein when they were expressed as PH-PTB⅐GST fusion proteins. The simplest interpretation of these data are that the PH domain function (i.e. binding to PI 4,5-P 2 or a negatively charged protein) is critical to elicit the downstream functions. This conclusion is also supported by the Cells expressing the PH-PTB⅐iSH2 constructs were homogenized and subjected to differential fractionation to obtain cytosolic (C), soluble particulate (SP) and insoluble particulate (IP) fractions as described under "Experimental Procedures." The insoluble particulate and immunoprecipitates of cytosolic and solubilized particulate fractions were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting using anti-HA antibodies (A). The amounts of the different chimeric molecules in the subcellular fractions were quantified using NIH image program and are plotted in B as the ratio of the particulate (SPϩIP) to cytosolic fraction (C). Data shown are representative of two independent experiments. finding that a chimeric molecule with only the PH domain of IRS-1 was also capable of stimulating the activation of the endogenous Akt. The somewhat weaker activity of this chimera in comparison to the PH-PTB molecule is consistent with its lower expression and somewhat decreased amount of associated PI 3-kinase activity. Because the construct, which only contained the PTB domain was expressed at lower levels and therefore had lower associated PI 3-kinase activity, it is not possible to conclude from this construct alone whether the PTB domain alone would have been capable at higher levels to induce the activation of Akt. However, the finding that the PH-PTB domain chimeric molecules whose PH domain function were impaired by either single or triple point mutations were incapable of eliciting the activation of the Akt argue that the PTB domain alone would not be sufficient to elicit this response because the PTB domain function in these chimeric molecules is still intact.
A potential role of the PH domain of IRS-1 is to help localize the protein in the correct intracellular compartment. To test this hypothesis, we examined the subcellular localization of the wild-type and two mutant chimeric molecules by biochemical fractionation of the cells. The wild-type PH-PTB chimeric molecule was found to be in the particulate fraction to a much greater degree then the chimeric molecules containing the mutations in the PH domain. These results are consistent with the recent report that the IRS-1 molecule is at least partially present in a particulate fraction of the cell, possibly because of its association with the cytoskeletal complex (29) as well as with a recent report that the same mutant we have studied (R28C) blocks the insulin induced translocation of a IRS⅐GFP construct to the membrane (10). Thus, the PH domain may serve to localize the chimeric molecules in the subcellular localization required for the subsequent activation of Akt.
In conclusion, the present work presents the first evidence that a functional PH domain of the IRS-1 molecule is required for its ability to signal to downstream molecules such as activation of Akt. This requirement is presumably because of the ability of the PH domain of IRS-1 to direct the PI 3-kinase to the correct subcellular compartment. Compromising the function of the PH domain, for example in insulin-resistant states, could therefore both decrease the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor as well as to link to subsequent downstream targets (2).