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Endocrinology Vol. 139, No. 5 2392-2398
Copyright © 1998 by The Endocrine Society

ARTICLES

Guanosine Triphosphatase-Activating Protein-Associated Protein, But Not src-Associated Protein p68 in Mitosis, Is a Part of Insulin Signaling Complexes1

Chin K. Sung, Woo S. Choi and VÍctor Sanchez-Margalet

Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033

Address all correspondence and requests for reprints to: Chin K. Sung, Ph.D., Department of Physiology and Biophysics, University of Southern California School of Medicine, 1333 San Pablo Street, MMR 626, Los Angeles, California 90033. E-mail: csung{at}hsc.usc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin receptor, following insulin stimulation of cells, triggers formation of various signaling complexes. In rat HTC hepatoma cells overexpressing normal human insulin receptors (HTC-IR), p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) forms signaling complexes containing the insulin receptor, insulin receptor substrate 1 (IRS-1), guanosine triphosphatase-activating protein (GAP) and 60–70 kDa phosphotyrosine proteins (p60–70). In the present study, we demonstrate that p60–70 interacts directly with the p85 subunit via src homology 2 domain of the latter. Employing antibodies specific to two p85 isoforms, p85{alpha} and p85ß, we demonstrate that HTC-IR cells express both p85 isoforms, and these isoforms induce the formation of similar signaling complexes in response to insulin. p60–70, present in both {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates, is a GAP-associated protein, but is distinct from the p68 src-associated protein in mitosis (Sam68) by several criteria. These data suggest that 1) GAP-associated protein, but not Sam68, is a part of insulin signaling complexes; and 2) p85{alpha} and p85ß form similar, but distinct, insulin receptor signaling complexes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PHOSPHATIDYLINOSITOL-3-KINASE (PI3K) has been implicated, although its exact molecular mechanism is obscure, in various cellular functions, including mitogenesis (1), stimulus-secretion coupling (2), and insulin-stimulated glucose transport (3, 4, 5, 6, 7, 8, 9). PI3K is a heterodimeric enzyme that consists of a p110 catalytic subunit (10, 11) and a p85 regulatory subunit (12, 13, 14). The p85 subunit contains two src homology 2 (SH2) domains and one SH3 domain that interact with phosphorylated tyrosines in specific motifs (15) and proline-rich domains, respectively (16, 17). PI3K, via its SH domains, has been found to be associated with many, but not all, tyrosine kinases (18, 19), indicating specificity in the assembly of the multiprotein signaling complexes (10). Recently, two isoforms of the p85 subunit of PI3K, p85{alpha} and p85ß, have been cloned and have an overall amino acid identity of 62% (14).

The insulin receptor is a {alpha}2ß2 tetrameric glycoprotein in the plasma membrane (20, 21, 22). When insulin binds to the {alpha}-subunit, intrinsic tyrosine kinase of the ß-subunit is activated, autophosphorylates, and tyrosine phosphorylates cellular proteins (20, 21, 22). Insulin receptor substrate-1 (IRS-1) is a major cellular substrate for both the insulin receptor and related insulin like growth factor I receptor (23, 24). Multiple tyrosine phosphorylation of IRS-1 in various motifs renders IRS-1 to interact with various SH2 containing proteins, including the p85 regulatory subunit of PI3K (24, 25). The insulin receptor itself, via its C-terminal Tyr1322-Thr-His-Met motif after tyrosine phosphorylation, is also capable of interacting with the SH2 domain of the PI3K p85 subunit (26, 27, 28, 29).

After insulin stimulation of cells, PI3K forms various signaling complexes with insulin receptor signaling proteins (20). They include the insulin receptor, IRS-1, and 60- to 70-kDa phosphoproteins. In the case of 60- to 70-kDa phosphoproteins (p60–70), there has been growing interest among investigators to identify and characterize these proteins (30, 31, 32, 33, 34). Employing {alpha}-guanosine triphosphatase (GTPase)-activating protein (GAP) antibody and {alpha}-p62 antibody, we have previously identified one of these proteins as the p62 GAP-associated protein (33). Later, this {alpha}-p62 was also reported to react with p68 src-associated protein in mitosis (Sam68) (35, 36). Thus, the question arises of whether p60–70 present in insulin receptor signaling complexes is related to Sam68.

To study the identity of p60–70 in {alpha}-p85 signaling complexes, we have employed a new antibody to Sam68 that does not cross-react with GAP-associated protein. We have also employed antibodies specific to p85{alpha} and p85ß to study the roles of p85 isoforms in the formation of insulin receptor signaling complexes. Here, we present data that p60–70 present in both {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates is indeed related to GAP-associated protein, but is distinct from Sam68. Two different p85 isoforms induce the formation of similar, but distinct, signaling complexes in response to insulin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents
Antibodies to phosphotyrosine ({alpha}-PY), rasGAP ({alpha}-GAP), and IRS-1 ({alpha}-IRS-1) and antiserum to the p85 regulatory subunit of PI3K ({alpha}-p85) were purchased from Upstate Biotechnology (Lake Placid, NY). {alpha}-p110, {alpha}-p62, and {alpha}-Sam68 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to p85{alpha} and p85ß ({alpha}-p85{alpha} and {alpha}-p85ß) were gifts from Dr. M. Waterfield (Ludwig Institute for Cancer Research, London, UK) (14, 37) and Dr. M. Czech (University of Massachusetts, Worcester, MA) (38), respectively. These antibodies were raised against the C-terminal 12-amino acid peptide of p85{alpha} and the C-terminal 15-amino acid peptide of p85ß, respectively. {alpha}-p85ß was received as a rabbit serum and was purified by protein G affinity chromatography before use. Rabbit {alpha}-IRS-1 antiserum was a gift from Dr. A. Maassen (University of Leiden, Leiden, The Netherlands). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), unless specified otherwise. Maltose-binding protein (MBP) fusion protein containing both SH2 domains of p85{alpha} (MBP-p85{alpha}-SH2) was prepared, bound to amylose resin beads (New England Biolabs, Beverley, MA), and used for the study as previously described (29).

Cells and preparation of soluble cell lysates
Rat HTC hepatoma cells overexpressing human insulin receptor (HTC-IR) were prepared and maintained as previously described (39). For experiments, cells were grown in 100-mm dishes to 90% confluence and serum starved for 16 h. They were treated for 5 min at 37 C with 100 nM insulin and solubilized for 30 min at 4 C in lysis buffer containing 20 mM Tris (pH 8.0), 1% Nonidet P-40, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol (DTT), 10% glycerol, 1 mM phenylmethylsulfonylfluoride, and 0.4 mM sodium orthovanadate (33). After centrifugation, the soluble cell lysates were used for the study.

Immunoprecipitation and Western blotting analysis
Soluble cell lysates (2 mg protein) were incubated with the appropriate antibodies for 3 h at 4 C and then with 50 µl protein A-Sepharose (1:1 suspension; Pharmacia Biotech, Piscataway, NJ) for 2 h at 4 C, unless specified otherwise. The immunoprecipitates were washed three times with lysis buffer. Fifty microliters of SDS-stop buffer containing 100 mM DTT were added to the immunoprecipitates and heated for 5 min at 95 C. These (15 µl each) were next resolved by Western blotting analysis with appropriate antibodies and Pierce enhanced chemiluminescence reagents (Pierce Chemical Co., Rockford, IL) (33).

In some cases, polyvinylidene difluoride (PVDF) membranes were treated with stripping solution [62.5 mM Tris (pH 6.7), 100 mM ß-mercaptoethanol, and 2% SDS] for 30 min at 70 C and reprobed with the appropriate antibodies.

Interaction of p60–70 with MBP fused to SH2 domains of p85{alpha}, MBP-p85-SH2
To study a direct protein-protein interaction, two methods were employed. In the first method, HTC-IR cell lysates (2 mg each) were initially denatured by heating for 10 min at 70 C in 4% SDS plus 10 mM DTT to disrupt endogenous protein-protein association and were next diluted 25-fold in lysis buffer and incubated for 1 h at 4 C with 100 µl amylose resin conjugated with 5 µg MBP-p85-SH2. After centrifugation, affinity precipitates were washed three times with lysis buffer and boiled in 30 µl SDS-stop buffer. These were next resolved by Western blotting analysis with {alpha}-PY.

To ascertain the direct interaction of p60–70 with MBP-p85-SH2, the second method, Far Western blotting analysis, was used. Briefly, {alpha}-p85 immunoprecipitates were resolved by SDS-PAGE and electrically transferred onto PVDF membrane, followed by incubation with 5% nonfat dry milk. The membrane was then incubated with either MBP or MBP-p85-SH2 (0.1 µg/ml) for 1 h and next with mouse {alpha}-MBP (0.1 µg/ml) for 1 h. After incubation for 20 min with {alpha}-mouse IgG conjugated with horseradish peroxidase (1:4000), the membrane was developed as described above.

In vitro tyrosine phosphorylation of GAP-associated protein, but not Sam68, by the insulin receptor
Insulin receptor was first prepared by wheat-germ agglutinin column chromatography (40). The purified insulin receptor (~200 fmol) was incubated for 1 h at 25 C in reaction buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 0.1% Triton X-100, 0.025% BSA, 1 mM phenylmethylsulfonylfluoride, 2 mM MnCl2, 10 mM MgCl2, and 100 nM insulin, followed by an additional 1-h incubation in the presence of 10 µM ATP (29). Next, this activated insulin receptor mixture was added to either {alpha}-p62 immunoprecipitates (2 mg cell lysates; 150 µg antibody; Fig. 4Go) or {alpha}-Sam68 immunoprecipitates (2 mg cell lysates; 2 µg antibody; Fig. 7aGo) prepared from unstimulated cells, and incubation was continued for 1 h. Reaction was stopped by the addition of SDS-stop buffer and heating for 5 min at 95 C. They were next analyzed by Western blotting with {alpha}-PY.



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  		Figure 4. In vitro tyrosine phosphorylation of GAP-associated protein by the activated insulin receptor. Wheat-germ-purified insulin receptor (~200 fmol) was first activated in vitro in the presence of insulin (100 nM) and ATP (10 µM). The activated insulin receptor was next added to {alpha}-p62 immunoprecipitates (2 mg unstimulated cell lysates with 150 µg {alpha}-p62), and incubation was continued for 1 h. These reaction mixtures were next resolved by Western blotting analysis with {alpha}-PY (1:10,000), as described in Materials and Methods. A representative of three experiments is shown.

 


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  		Figure 7. No direct tyrosine phosphorylation of Sam68 by the activated insulin receptor. Insulin receptor was prepared and activated as described in Fig. 4Go. A, The activated insulin receptor was added to immunoprecipitates of {alpha}-Sam68 (2 µg), normal rabbit IgG (NRG; 2 µg), and {alpha}-IRS-1 antiserum (20 µl) prepared from unstimulated cell lysates (2 mg each) and processed as described in Fig. 4Go. B, A recombinant IRS-1 was used as a substrate for the insulin receptor. A representative of three experiments is shown.

 
Depletion of Sam68 by poly(U)-Sepharose
HTC-IR cell lysates (2 mg), prepared as described above, were incubated with either 100 µl poly(U)-Sepharose or control Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) for 3 h at 4 C and centrifuged. The pellets were washed three times in lysis buffer and resuspended in SDS-stop buffer for Western blotting analysis. After the second precipitation of the supernatants with poly(U)-Sepharose, the resultant supernatants were next immunoprecipitated with {alpha}-p85{alpha} and {alpha}-p85ß as described above. Poly(U)-Sepharose precipitates and immunoprecipitates were then analyzed by Western blotting with {alpha}-PY.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-PI3K ({alpha}-p85 and {alpha}-p110)
HTC-IR cells were incubated with 100 nM insulin for 5 min and solubilized. Cell lysates were then immunoprecipitated with {alpha}-p85, subjected to SDS-PAGE, and resolved by Western blotting analysis with {alpha}-PY. Three major tyrosine-phosphorylated proteins were observed (Fig. 1AGo). They were previously identified as the insulin receptor ß-subunit, IRS-1, and GAP-associated protein as one of p60–70 phosphoproteins (33). When cell lysates were immunoprecipitated with {alpha}-p110, the similar, but distinct in intensities of phosphoproteins, pattern of phosphotyrosine proteins was obtained as that in {alpha}-p85 immunoprecipitates (Fig. 1AGo).



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  		Figure 1. A, Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-PI3K. HTC-IR cells were incubated with 100 nM insulin for 5 min and solubilized. Cell lysates (2 mg) were immunoprecipitated with {alpha}-p85 antiserum (2 µl) and {alpha}-p110 (2 µg), subjected to SDS-PAGE, and analyzed by Western blotting with {alpha}-PY, as described in Materials and Methods. A representative of four independent experiments is shown. IR-ß, Insulin receptor ß-subunit; IP-AB, immunoprecipitating antibody; WB-AB; Western blotting antibody. B, Affinity precipitation of phosphotyrosine-containing proteins with MBP-p85-SH2 fusion protein after insulin treatment of cells. Cell lysates were first prepared and denatured to disrupt endogenous protein-protein interactions. These denatured cell lysates were then incubated with MBP-p85-SH2 fusion protein, followed by subsequent Western blotting analysis with {alpha}-PY. A representative of three experiments is shown. C, Far Western blotting analysis of {alpha}-p85 immunoprecipitates with MBP-p85-SH2 fusion protein. {alpha}-p85 immunoprecipitates were first resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was then incubated with either MBP or MBP-p85-SH2 fusion protein, followed by incubation with mouse {alpha}-MBP and {alpha}-mouse IgG conjugated with horseradish peroxidase. Enhanced chemiluminescence was next performed similarly, as described for the Western blotting analysis. A representative of three experiments is shown.

 
Interaction of p60–70 with MBP-p85-SH2 fusion protein
High affinity interaction between tyrosine-phosphorylated protein and a specific SH2 domain has been well established (41). To study whether interaction of p60–70 with the p85 subunit of PI3K is mediated by SH2 domains of p85, we first prepared cell lysates after insulin stimulation of cells and denatured them to disrupt endogenous protein-protein complexes. These denatured cell lysates were next incubated with amylose resin prebound to MBP fusion protein containing both SH2 domains of p85{alpha} (MBP-p85-SH2) and centrifuged, followed by subsequent Western blotting analysis with {alpha}-PY (Fig. 1BGo). In these affinity precipitates, a similar pattern of tyrosine-phosphorylated proteins was obtained as those in {alpha}-p85 and {alpha}-p110 immunoprecipitates of cell lysates (Fig. 1AGo). Plain amylose resin did not precipitate any of these proteins (data not shown). Direct associations of both the insulin receptor and IRS-1 with SH2 domains of p85 have been previously demonstrated (24, 27, 29). The present data suggest that p60–70 may also directly interact with p85 via SH2 domains of p85. One potential problem with this approach is that one cannot exclude the possibility of incomplete denaturation of cell lysates and/or reformation of protein complexes. Thus, one can argue against the direct interaction of p60–70 with MBP-p85-SH2.

To ascertain the direct protein-protein interaction between p60–70 and p85, Far Western blotting analysis was performed. In this method, proteins coimmunoprecipitated with {alpha}-p85 were first separated on a membrane and then incubated with protein, either MBP as a control or MBP-p85-SH2 fusion protein. Subsequent steps were similar to those in Western blotting analysis. In the membrane incubated with MBP-p85-SH2 fusion protein, both IRS-1 and p60–70, but not the insulin receptor ß-subunit, were identified (Fig. 1CGo). These data clearly indicate that both p60–70 and IRS-1 directly interacted with p85 via SH2 domains of the latter. It was interesting to find that the insulin receptor was not detected under the conditions employed. This may be explained by the fact that only a small fraction of the insulin receptor directly interacts with p85 (24, 27).

Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-p85{alpha} and {alpha}-p85ß
To study whether both p85{alpha} and p85ß form similar insulin receptor signaling complexes, we prepared HTC-IR cell lysates as described above. Cell lysates were then immunoprecipitated with {alpha}-p85{alpha} (mouse IgG) and {alpha}-p85ß (rabbit IgG), subjected to SDS-PAGE, and resolved by Western blotting analysis with {alpha}-PY. These immunoprecipitates revealed a similar, but distinct, pattern of phosphotyrosine-containing proteins (Fig. 2Go). In {alpha}-p85ß immunoprecipitates, there were more insulin receptor and less p60–70 coimmunoprecipitated than in {alpha}-p85{alpha} immunoprecipitates. These data are consistent with the previous finding that both p85 isoforms associate with IRS-1 (42). Control IgGs (normal mouse IgG and normal rabbit IgG) did not coimmunoprecipitate any of these tyrosine-phosphorylated proteins.



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  		Figure 2. Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-p85{alpha} and {alpha}-p85ß. HTC-IR cells were incubated for 5 min with 100 nM insulin and solubilized. Cell lysates (2 mg) were immunoprecipitated with {alpha}-p85{alpha} (20 µl cultured supernatant), {alpha}-p85ß (10 µg), normal mouse IgG (NMG; 10 µg), and normal rabbit IgG (NRG; 10 µg); subjected to SDS-PAGE; and analyzed by Western blotting with {alpha}-PY (1:10,000). A representative of several experiments is shown. IR-ß, Insulin receptor ß-subunit; IP-AB, immunoprecipitating antibody; WB-AB; Western blotting antibody. A band at approximately 50 kDa is a heavy chain of IgG.

 
Coimmunoprecipitation of GAP-associated protein with {alpha}-p85ß and {alpha}-p85{alpha}
We previously demonstrated that a p60–70 coimmunoprecipitated with {alpha}-p85 (that reacts with both p85{alpha} and p85ß) was the GAP-associated protein by several criteria (33). To study whether both {alpha}-p85{alpha} and {alpha}-p85ß coimmunoprecipitated GAP-associated protein, we prepared various immunoprecipitates ({alpha}-p85ß, {alpha}-p85{alpha}, and {alpha}-GAP) and analyzed them by Western blotting with {alpha}-p62 (Fig. 3Go). In {alpha}-p85ß immunoprecipitates, there was little p62 present in the basal state. After insulin treatment of cells, the p62 band was clearly visible. Similar results were obtained with {alpha}-GAP immunoprecipitates. In {alpha}-p85{alpha} immunoprecipitates, however, there was a faint p62 band present in the basal state and no significant increase in intensity of this p62 band after insulin treatment of cells. In the control experiment in which normal rabbit IgG (instead of rabbit {alpha}-p62) was used to probe the same membrane, there was no p62 detected (data not shown). These data suggest that GAP-associated protein is present in {alpha}-p85ß immunoprecipitates, but little is present in {alpha}-p85{alpha} immunoprecipitates. Insulin treatment of cells increased the association of p62 only with p85ß (as well as with GAP), but not with p85{alpha}.



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  		Figure 3. Increase in GAP-associated protein coimmunoprecipitated with {alpha}-p85ß and {alpha}-GAP, but not with {alpha}-p85{alpha}. Cell lysates, prepared as described in Fig. 1Go, were immunoprecipitated with {alpha}-p85ß (10 µg), {alpha}-p85{alpha} (20 µl cultured supernatant), and {alpha}-GAP (20 µg) and Western blotted with {alpha}-p62 (1:2500). A representative of several experiments is shown.

 
Direct tyrosine phosphorylation of the GAP-associated protein by the activated insulin receptor in vitro
To study whether GAP-associated protein, one of p60–70 present in {alpha}-p85 immunoprecipitates, is a direct substrate of the insulin receptor, we immunoprecipitated unstimulated cell lysates using {alpha}-p62. As {alpha}-p62 does not effectively react with the native form of p62, cell lysates were denatured by SDS and heating before immunoprecipitation. The {alpha}-p62 immunoprecipitates were next incubated with the activated insulin receptor, followed by Western blotting analysis with {alpha}-PY (Fig. 4Go). Activation of the insulin receptor was demonstrated by increased tyrosine phosphorylation of the insulin receptor ß-subunit (lanes 1 and 2, Fig. 4Go). {alpha}-p62 immunoprecipitates alone showed no tyrosine phosphorylation of p60–70 (lane 3, Fig. 4Go). When {alpha}-p62 immunoprecipitates and the activated insulin receptor were incubated together, there was an increase in tyrosine phosphorylation of p62 (lanes 4 and 5, Fig. 4Go). The identity of the additional band below p62 is unclear at the present time. The data suggest that the GAP-associated protein serves as a direct substrate of the insulin receptor in vitro.

Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-p85{alpha}, {alpha}-p85ß, {alpha}-IRS-1, {alpha}-GAP, and {alpha}-Sam68
As mentioned previously, one of the p60–70s in {alpha}-p85 immunoprecipitates was identified as a GAP-associated protein by various criteria (Fig. 3Go) (33). Recently, it was reported that the {alpha}-p62 used in our experiments recognized both GAP-associated protein and Sam68 (35, 36). To study whether p60–70 present in {alpha}-p85 immunoprecipitates was related to Sam68, we employed a new antibody specific to Sam68 that does not recognize GAP-associated protein. Here, we present three independent results confirming that the p60–70 in {alpha}-p85 immunoprecipitates is not Sam68 ( Figs. 5–7GoGoGo).



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  		Figure 5. A, Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-p85{alpha}, {alpha}-p85ß, {alpha}-IRS-1, {alpha}-GAP, and {alpha}-Sam68. Cell lysates, prepared as described in Fig. 1Go, were immunoprecipitated with various antibodies: {alpha}-p85{alpha} (20 µl cultured supernatant), {alpha}-p85ß (10 µg), {alpha}-IRS-1 antiserum (20 µl), {alpha}-GAP (20 µg), and {alpha}-Sam68 (2 µg). These immunoprecipitates were next resolved by Western blotting analysis with {alpha}-PY (1:10,000), as described in Fig. 1Go. A representative of three experiments is shown. B, Identification of Sam68 only in {alpha}-Sam68 immunoprecipitates. The PVDF membrane shown in a was stripped off and reprobed with {alpha}-Sam68 (1:1,000). A representative of three experiments is shown.

 


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  		Figure 6. Coimmunoprecipitation of phosphotyrosine-containing proteins with {alpha}-p85{alpha} and {alpha}-p85ß after poly(U)-Sepharose pretreatment of cell lysates. Cell lysates (2 mg each) were pretreated twice with poly(U)-Sepharose (100 µl suspension) to deplete Sam68, as described in Materials and Methods (lanes 5–8). The resultant supernatants were then immunoprecipitated with {alpha}-p85{alpha} (20 µl cultured supernatant; lanes 9 and 10) and {alpha}-p85ß (10 µg; lanes 11 and 12). For initial immunoprecipitation with {alpha}-p85{alpha} (lanes 1 and 2) and {alpha}-p85ß (lanes 3 and 4), cell lysates were treated in parallel with plain Sepharose 4B. Poly(U)-Sepharose precipitates and immunoprecipitates were next analyzed by Western blotting with {alpha}-PY (1:10,000). A representative of several experiments is shown.

 
Cell lysates were immunoprecipitated with a panel of antibodies, including {alpha}-p85{alpha}, {alpha}-p85ß, {alpha}-IRS-1, {alpha}-GAP, and {alpha}-Sam68. They were next resolved by Western blotting analysis with {alpha}-PY (Fig. 5AGo). In {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates, there were three major tyrosine-phosphorylated proteins, as expected. As previously reported, {alpha}-IRS-1 immunoprecipitates contained IRS-1 and insulin receptor, but not p60–70, and {alpha}-GAP immunoprecipitates contained the insulin receptor and p60–70, but not IRS-1. In {alpha}-Sam68 immunoprecipitates, however, there was no phosphoprotein detected after insulin stimulation of cells. When the same PVDF membrane was stripped and reprobed with {alpha}-Sam68, only {alpha}-Sam68 immunoprecipitates revealed Sam68 (Fig. 5BGo). The {alpha}-IRS-1 used in this experiment was a rabbit antiserum, and this may account for the high background. These data suggest that Sam68 is not tyrosine phosphorylated by insulin treatment of cells and does not form a complex with PI3K in response to insulin.

Depletion of Sam68 by poly(U)-Sepharose pretreatment of cell lysates
The cloned Sam68 (originally thought to be p62 GAP-associated protein) has a strong regional homology to a putative heteronuclear ribonuclear particle protein and binds to RNA (43). To further prove that p60–70 present in {alpha}-p85 immunoprecipitates is not Sam68, we first depleted Sam68 by preincubation of cell lysates with poly(U)-Sepharose twice. These Sam68-depleted cell lysates were next immunoprecipitated with either {alpha}-p85{alpha} or {alpha}-p85ß. For initial {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates, we pretreated cell lysates twice with control Sepharose and performed subsequent immunoprecipitation in parallel. Poly(U)-Sepharose precipitates and immunoprecipitates were then analyzed by Western blotting with {alpha}-PY (Fig. 6Go).

The first poly(U)-Sepharose treatment (lanes 5 and 6, Fig. 6Go) precipitated many phosphoproteins from the cell lysates, and double poly(U)-Sepharose treatments were sufficient to deplete most phosphoproteins bound to poly(U)-Sepharose (lanes 7 and 8, Fig. 6Go). The initial {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates revealed three major phosphoproteins, as expected (lanes 1–4, Fig. 6Go). {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates of poly(U)-Sepharose-pretreated cell lysates showed a similar pattern of phosphorylated proteins with similar band intensities (lanes 9–12, Fig. 6Go). It should be noted that in poly(U)-Sepharose precipitates (lanes 5 and 6), a phosphoprotein migrated at a slightly lower position than the p60–70 present in {alpha}-p85 immunoprecipitates. The identity of this protein is unknown. These data suggest that p60–70 present in {alpha}-p85 immunoprecipitates are not depleted by poly(U)-Sepharose pretreatment and are distinct from Sam68.

Sam68 is not directly tyrosine phosphorylated by the activated insulin receptor in vitro
To confirm that Sam68 is not tyrosine phosphorylated by the insulin receptor, we performed a similar in vitro phosphorylation experiment with {alpha}-Sam68 immunoprecipitates, as shown in Fig. 4Go. Again, insulin receptor activation was demonstrated by increased tyrosine phosphorylation of the insulin receptor ß-subunit (lanes 1 and 2, Fig. 7AGo). When the activated insulin receptor and {alpha}-Sam68 immunoprecipitates were incubated together, there was no phosphorylation of Sam68 (lanes 3 and 4, Fig. 7AGo). In this experiment, normal rabbit IgG immunoprecipitates were used as a negative control for {alpha}-Sam68 immunoprecipitates (lanes 5 and 6, Fig. 7AGo). In {alpha}-IRS-1 immunoprecipitates used as a positive control, IRS-1 was tyrosine phosphorylated by the activated insulin receptor (lanes 7 and 8, Fig. 7AGo). When a recombinant IRS-1 was used as a substrate, IRS-1 was more effectively tyrosine phosphorylated under the same assay conditions (Fig. 7BGo). These data suggest that only GAP-associated protein, not Sam68, serves as a direct substrate of the insulin receptor in vitro. It is possible, however, that phosphorylation of Sam68 by the insulin receptor was too low to be detected under the conditions used.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have demonstrated that rat HTC hepatoma cells express two isoforms of p85, p85{alpha} and p85ß, and these isoforms form similar, but distinct, signaling complexes in response to insulin. Both p85 isoforms are capable of forming complexes with three major tyrosine-phosphorylated proteins, including the insulin receptor, IRS-1, and p60–70. p60–70, like the insulin receptor and IRS-1, interacts directly with the fusion protein-containing SH2 domains of p85. p60–70 present in {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates is related to GAP-associated protein, but is distinct from Sam68 by various criteria. Although GAP-associated protein is present in both {alpha}-p85ß immunoprecipitates and {alpha}-p85{alpha} immunoprecipitates (little, if any), only {alpha}-p85ß immunoprecipitates, like {alpha}-GAP immunoprecipitates, showed an increase in p62 after insulin stimulation of cells.

PI3K has been reported to play a crucial role in many biological functions of insulin, including stimulation of glucose transport, amino acid transport, DNA synthesis, and p70 ribosomal S6 protein kinase (20, 44). Many receptor and nonreceptor tyrosine kinases stimulate PI3K, but not all of these processes lead to stimulation of insulin action such as glucose transport (1, 2, 45, 46). Thus, the question arises: where does the specificity for insulin action come? This has led many investigators to look for specific signaling proteins that are associated with PI3K after insulin stimulation of cells.

In our previous studies and those by others, one or more of p60–70 tyrosine-phosphorylated proteins have been identified in {alpha}-p85 immunoprecipitates after insulin stimulation of cells such as rat primary adipocytes and HTC-IR cells (30, 31, 32, 33, 34). The protein(s) is tyrosine phosphorylated in an insulin dose- and time-dependent manner. Previously, we have identified in HTC-IR cells one of these proteins as GAP-associated protein by three criteria: 1) depletion of p60–70 in subsequent {alpha}-GAP immunoprecipitates after {alpha}-p85 immunoprecipitation, 2) Western blotting analysis of p60–70 with {alpha}-p62, and 3) increase in GAP in {alpha}-p85 immunoprecipitates after insulin stimulation of cells (33). It is unclear whether a similar GAP-associated protein is present in rat primary adipocytes.

It should be noted that the intensity of tyrosine phosphorylation of this p62 in {alpha}-GAP immunoprecipitates was much lower than that of p60–70 in {alpha}-p85 immunoprecipitates (33). This observation led us to propose that there were other p60–70 present in {alpha}-p85 immunoprecipitates and was consistent with the following several reports. Firstly, Hosomi et al. (30) reported, based on phosphopeptide map analysis, that the p60–70 coimmunoprecipitated with {alpha}-p85 is different from the p60–70 coimmunoprecipitated with {alpha}-GAP. Secondly, a different p60 that is not recognized by {alpha}-p62 was reported as a major GAP-associated protein in NIH 3T3 cells (47). Lastly, Lavan et al. recently reported in primary adipocytes that p60 associated with p85 of PI3K is IRS-3, which is similar to IRS-1/2 (48).

Recently, human p62 complementary DNA, which was originally thought to encode p62 GAP-associated protein (43), was found to encode Sam68 (49). Moreover, the {alpha}-p62 used in our past and present studies was generated against amino acids 103–281 of what turned out to be Sam68, not p62 GAP-associated protein. These reports made it necessary to reevaluate the identity of p60–70 present in {alpha}-p85 immunoprecipitates. First, we employed a new antibody against amino acids 331–443 of Sam68 that reacts only with Sam68, but not with GAP-associated protein. Immunoprecipitation and Western blotting analysis clearly demonstrated that Sam68 was not tyrosine phosphorylated by insulin treatment of cells and did not form complexes with PI3K. Next, we used the ability of Sam68 to bind RNA to further prove that Sam68 is not a part of the insulin receptor signaling complex. Pretreatment of cell lysates twice with poly(U)-Sepharose to deplete Sam68 (43) did not affect the intensity of tyrosine phosphorylation of p60–70 in subsequent {alpha}-p85{alpha} and {alpha}-p85ß immunoprecipitates. Lastly, an in vitro phosphorylation study showed that Sam68 in {alpha}-Sam68 immunoprecipitates was not tyrosine phosphorylated by the insulin receptor in vitro. It should be noted that only the protein immunoprecipitated with {alpha}-p62, but not with that immunoprecipitated with {alpha}-Sam68, was directly phosphorylated by the insulin receptor, although both antibodies were generated against peptides of Sam68. Moreover, {alpha}-p62, but not {alpha}-Sam68, reacted with p60–70 present in {alpha}-GAP immunoprecipitates. Others have also reported that the {alpha}-p62 reacted with GAP-associated protein (35, 36). Taken together, it is clear that the p60–70 present in {alpha}-p85 immunoprecipitates was not Sam68 and was indeed a GAP-associated protein. Currently, we are using two-dimensional gel electrophoresis to further separate these proteins. Whether the GAP-associated protein present in {alpha}-p85 immunoprecipitates is related to the recently cloned p62 GAP-associated protein, p60dok, awaits further investigation (50, 51).

p60–70 present in {alpha}-p85ß and {alpha}-GAP immunoprecipitates contained GAP-associated protein whose amount was increased by insulin stimulation of cells. These data suggest that insulin treatment of cells increased tyrosine phosphorylation of a GAP-associated protein and increased association of this protein with p85ß and GAP.

It should also be noted that in {alpha}-p85 immunoprecipitates, there was another phosphoprotein at 110–120 kDa after insulin treatment of cells. In liver, a membrane glycoprotein pp120 has been reported to be tyrosine phosphorylated by insulin treatment and associated with the insulin internalization rate (52, 53). In 3T3-L1 adipocytes, a 115-kDa protein has been reported to be tyrosine phosphorylated by insulin and associated with SHPTP2 (54). Further study is necessary to determine whether our protein is related to these two reported proteins.

Taken together, these data suggest that a GAP-associated protein, but not Sam68, is a part of insulin signaling complex, and both p85{alpha} and p85ß isoforms trigger formation of similar, but distinct, signaling complexes after insulin stimulation of cells.


   Acknowledgments
 
We thank Dr. M. Waterfield at Ludwig Institute for Cancer Research for {alpha}-p85{alpha}, Drs. K. Baltensberger and M. Czech at the University of Massachusetts for {alpha}-p85ß, and Dr. A. Maassen for {alpha}-IRS-1.


   Footnotes
 
1 This work was supported by NIH Grant DK-51015, the American Diabetes Association, and a Zumberge fellowship at the University of Southern California. Back

Received December 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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