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J Biol Chem, Vol. 274, Issue 39, 27969-27974, September 24, 1999


JAK1-dependent Phosphorylation of Insulin Receptor Substrate-1 (IRS-1) Is Inhibited by IRS-1 Serine Phosphorylation*

Keith A. Cengel and Gregory G. FreundDagger

From the Departments of Pathology and Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) reduces its ability to act as an insulin receptor substrate and inhibits insulin receptor signal transduction. Here, we report that serine phosphorylation of IRS-1 induced by either okadaic acid (OA) or chronic insulin stimulation prevents interferon-alpha (IFN-alpha )-dependent IRS-1 tyrosine phosphorylation and IFN-alpha -dependent IRS-1/phosphatidylinositol 3'-kinase (PI3K) association. In addition, we demonstrate that serine phosphorylation of IRS-1 renders it a poorer substrate for JAK1 (Janus kinase-1). We found that treatment of U266 cells with OA induced serine phosphorylation of IRS-1 and completely blocked IFN-alpha -dependent tyrosine phosphorylation of IRS-1 and IFN-alpha -dependent IRS-1/PI3K association. Additionally, IRS-1 from OA-treated cells could not be phosphorylated in vitro by IFN-alpha -activated JAK1. Chronic treatment of U266 cells with insulin led to a 50% reduction in IFN-alpha -dependent tyrosine phosphorylation of IRS-1 and IRS-1/PI3K association. More importantly, serine-phosphorylated IRS-1-(511-722) could not be phosphorylated in vitro by IFN-alpha -activated JAK1. Taken together, these data indicate that serine phosphorylation of IRS-1 prevents its subsequent tyrosine phosphorylation by JAK1 and suggest that IRS-1 serine phosphorylation may play a counter-regulatory role in pathways outside the insulin signaling system.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tyrosine phosphorylation of IRS-11 is common to the signal transduction pathways of a variety of growth factors, hormones, and cytokines (1). Since the first description of IRS-1 tyrosine phosphorylation in insulin-stimulated Fao hepatoma cells (2), IRS-1 phosphorylation has been shown to occur after insulin-like growth factor-1, IL-2, IL-4, IL-10, IL-15, growth hormone, leukemia inhibitory factor, and oncostatin M stimulation (3-6). Recently, type I interferons were demonstrated to induce tyrosine phosphorylation of IRS-1 (7-9). This class of interferons is composed of alpha -, beta -, and omega -subtypes, which all activate the type I IFN receptor (10). After IFN-alpha binds to the type I IFN receptor, it induces receptor oligomerization, which, in turn, activates the Janus kinase family members Tyk2 and JAK1 (11-17). Activation of JAK1 induces IFN receptor tyrosine phosphorylation and leads to subsequent JAK1-dependent tyrosine phosphorylation of STAT1, STAT2, STAT3, IRS-1, and IRS-2 (6, 10) and the association of STAT3, IRS-1, and IRS-2 with PI3K (7-9, 18).

Tyrosine-phosphorylated IRS-1 coordinates the intracellular signaling of various growth factor, hormone, and cytokine receptors in part by binding to SH2 domain-containing proteins (1). The association of PI3K with IRS-1 is the best characterized of these IRS-1/SH2 domain interactions and, in signaling pathways that require IRS-1, appears to be a critical step in effecting post-receptor function (1, 19-22). PI3K is a heterodimeric protein composed of a regulatory 85-kDa subunit (p85) and a catalytic 110-kDa subunit (p110) (19). p85 contains N- and C-terminal SH2 domains that bind to tyrosine-phosphorylated IRS-1 and induce PI3K activation (20-22). p110 phosphorylates phosphoinositides on the D-3 position of the inositol ring, generating 3,4-bis- and 3,4,5-trisphosphates (23, 24). In the IFN-alpha signaling cascade, PI3K is recruited to and activated by tyrosine-phosphorylated IRS-1 (7-9, 18). Inhibition of PI3K activity with wortmannin leads to decreased IFN-alpha -dependent STAT3 serine phosphorylation and decreased IFN-alpha -dependent transcriptional activation (18).

Growth factors, hormones, and cytokines can also induce serine phosphorylation of IRS-1 (3, 4, 25-28), and in the insulin signaling system, serine phosphorylation of IRS-1 blocks insulin action (26-35). Treatment of 3T3-L1 adipocytes with the serine phosphatase inhibitor okadaic acid (OA) results in increased IRS-1 serine phosphorylation, reduced IRS-1/insulin receptor association, and decreased insulin receptor-dependent tyrosine phosphorylation of IRS-1 (26). Similarly, IRS-1 serine phosphorylation induced by phorbol esters, tumor necrosis factor-alpha , platelet-derived growth factor, angiotensin II, PI3Kassociated serine kinase (PAS kinase), and insulin reduces subsequent insulin receptor-dependent IRS-1 tyrosine phosphorylation and IRS-1/PI3K association (27-35). Although much is known about the effect of IRS-1 serine phosphorylation on insulin signaling, nothing is known about the impact of IRS-1 serine phosphorylation on cytokine signaling. Here, we report that serine phosphorylation of IRS-1 induced by either OA or chronic insulin stimulation inhibits IFN-alpha -dependent tyrosine phosphorylation of IRS-1 by JAK1 and blocks subsequent IRS-1/PI3K association. These findings indicate that IRS-1 serine phosphorylation may play a counter-regulatory role in signaling pathways outside the insulin system and suggest that hyperinsulinemia may alter signaling of JAK1- dependent cytokine receptors.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The myeloma cell line U266 was purchased from American Type Culture Collection (Manassas, VA). [gamma -32P]ATP and 32Pi were purchased from Amersham Pharmacia Biotech. Anti-PI3K p85 (catalog no. 06-195), anti-phosphotyrosine (catalog no. 05-321), and anti-IRS-1 (catalog no. 06-248C) antisera were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-JAK1 antiserum (catalog no. 15206E) was purchased from Pharmingen (San Diego, CA). Neonatal bovine serum was purchased from Biocell (Rancho Dominguez, CA). Protein G-Sepharose, glutathione-Sepharose, Precission protease, and pGex6P3 vector were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Recombinant human IFN-alpha -1 was purchased from Intergen Co. (Purchase, NY). Polyvinylidene difluoride membrane was purchased from Bio-Rad. Cellulose-coated TLC plates were purchased from Analtech Inc. (Newark, DE). Minifilter columns (catalog no. QSQ) were purchased from Midwest Scientific (Valley Park, MO). All other cell culture reagents and chemicals were purchased from Sigma. Oligonucleotide primers were purchased from Operon Technologies, Inc. (Alameda, CA). All other molecular biology reagents and chemicals were purchased from Promega.

Cell Culture-- U266 cells were grown in growth medium (RPMI 1640 medium supplemented with 10% neonatal bovine serum, 2.0 g/liter glucose, 100,000 units/liter penicillin, and 100 mg/liter streptomycin). Cells were passaged 1:1 with fresh medium every 3 days. For OA treatment, cells were washed twice and resuspended in growth medium supplemented with 1 µM OA. For insulin treatment, cells were washed twice and resuspended in growth medium supplemented with 1 nM insulin.

PI3K Assays-- PI3K assays were performed as described previously (36). In brief, 20 × 106 cells/ml were treated as indicated and lysed in 1 ml of ice-cold lysis buffer (1% Nonidet P-40, 100 mM NaCl, 50 mM NaF, 1 mM dithiothreitol, 25 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 mM sodium orthovanadate, 250 nM okadaic acid, and 50 mM Tris, pH 7.4). IRS-1 was immunoprecipitated from lysates with 4 µl of anti-IRS-1 antiserum/test, and the resultant immune complexes were washed extensively. Kinase reactions were performed in 100 µl of buffer containing 0.33 mg/ml L-alpha -phosphatidylinositol, 7.5 mM MgCl2, 0.4 mM EGTA, 0.4 mM NaPO4, 7.5 µM [gamma -32P]ATP (13 µCi/nmol), and 20 mM HEPES, pH 7.1, at 22 °C for 15 min. The assay conditions used were linear with respect to time and amount of kinase. Phospholipids were extracted with 1:1 chloroform/methanol and resolved on silica gel plates by TLC in chloroform/methanol/4 M ammonium hydroxide (75:58:17). Results were analyzed by autoradiography on a Molecular Dynamics PhosphorImager system.

Western Analysis-- Western analysis was performed as described previously (37). In brief, 20 × 106 cells/ml were treated as indicated and lysed in 1 ml of ice-cold lysis buffer. Proteins of interest were immunoprecipitated with the indicated antiserum (4 µl/test), and the resultant immune complexes were washed extensively. Proteins were resolved by SDS-PAGE under reducing conditions on 10% gels, electrotransferred to polyvinylidene difluoride membrane, and probed with the indicated antiserum. Immunoreactive proteins were visualized by secondary detection using an 125I-labeled goat anti-rabbit antibody, followed by autoradiography and densitometry.

JAK1 Kinase Assays-- Cells (20 × 106/ml) were treated as indicated and lysed in 1 ml of ice-cold lysis buffer. JAK1 was immunoprecipitated from lysates with 4 µl of anti-JAK1 antiserum/test, and the resultant immune complexes were washed extensively. Kinase reactions were performed in 100 µl of buffer containing 7.5 mM MgCl2, 2.5 mM MnCl2, 20 µM [gamma -32P]ATP (10 µCi/nmol), and 20 mM HEPES, pH 7.5, at 22 °C for 20 min with 5 µg/ml IRS-1-(511-772) or eluted IRS-1 or with no substrate as indicated. In reactions using eluted IRS-1 from U266 cells, IRS-1 was immunoprecipitated from 20 × 106 cells. The resultant immune complexes were eluted for 60 min at 37 °C in 10 µl of 100 mM dithiothreitol, 0.5% SDS, 1 mg/ml bovine serum, and 20 mM HEPES, pH 7.4, and used in kinase reactions at a 1:10 dilution. Kinase reactions were terminated by addition of SDS-PAGE loading buffer, and the assay conditions used were linear with respect to time and amount of kinase. Resultant phosphoproteins were resolved by SDS-PAGE under reducing conditions on 7-20% gradient gels. Serine and threonine phosphoamino acids were base-hydrolyzed (39), and phosphotyrosine-containing proteins were examined by autoradiography and densitometry.

IRS-1-(511-772) Expression-- The coding sequence for amino acids 511-772 of IRS-1 was amplified from rat IRS-1 sequence (a kind gift of Morris F. White) by previously described methods (34) using the forward primer 5'-CAGGATCCGATCTGGATAACCGGTTTC-3' and the reverse primer 5'-GAGAATTCGCGCTGGGTGTGCTAAAAG-3'. This 799-base pair product was introduced into the pGex6P3 plasmid using the BamHI and EcoRI restriction sites. GST-IRS-1-(511-772) was produced as described previously (38). In brief, protein expression was induced in Escherichia coli strain BL21 by addition of isopropyl-beta -D-thiogalactopyranoside to a final concentration of 1 mM. After 1 h, bacteria were lysed by mild sonication at 4 °C in phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) supplemented with 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 mM dithiothreitol. GST fusion proteins were affinity-purified from clarified lysates using glutathione-Sepharose, and GST was removed by digestion with 5 units of Precission protease at 4 °C.

Whole Cell Phosphorylation/Phosphoamino Acid Analysis-- Whole cell phosphorylation was performed as described previously (37). In brief, 20 × 106 cells were suspended in 1 ml of phosphate-free RPMI 1640 medium supplemented with 0.75 mCi/ml 32Pi and 20 mM HEPES, pH 7.4, at 37 °C for 1.5 h. For OA treatment, 1 µM OA was added at 1.5 h for 30 min. Cells were lysed, and IRS-1 was immunoprecipitated as described above. Phosphoproteins were resolved by SDS-PAGE under reducing conditions on 8% gels. For phosphoamino acid analysis, phosphoproteins were electrotransferred to polyvinylidene difluoride membrane. Bands of interest were excised and acid-hydrolyzed in 6 N constant boiling HCl for 2 h at 110 °C. Samples were treated with three cycles of water resuspension and evaporation and then resuspended in H2O/acetic acid/pyridine (89:10:1) running buffer containing 0.3 mg/ml phosphoserine, phosphothreonine, and phosphotyrosine standards. Phosphoamino acids were separated on cellulose-coated plates by high voltage TLC, and standards were visualized with ninhydrin. Results were analyzed by autoradiography and densitometry.

PAS Kinase Assays-- PAS kinase assays were performed as described previously (37). In brief, PAS kinase was purified from 20 × 106 cells by affinity chromatography using glutathione-Sepharose-bound GST-p85 protein. Kinase reactions were performed in 100 µl of reaction buffer containing 5 µg/ml IRS-1-(511-772), 0.4 mM EGTA, 0.4 mM NaPO4, 1 µM [gamma -32P]ATP (100 µCi/nmol), and 20 mM HEPES, pH 7.1, at 22 °C with or without 10 mM MgCl2 as a cofactor. For JAK1 kinase assays, IRS-1-(511-772) was prephosphorylated in the absence of [gamma -32P]ATP and recovered by filtration through minifilter columns. IFN-alpha -activated JAK1 was isolated as described above, and JAK1 kinase assays using prephosphorylated IRS-1-(511-772) as a substrate were performed as described above. Reactions were terminated using SDS-PAGE loading buffer and were linear with respect to time and amount of kinase. Phosphoproteins were resolved by SDS-PAGE under reducing conditions on 7-20% gradient gels and examined by autoradiography and densitometry.

Lysate Kinase Assays-- Cells (10 × 106/10 ml) were treated with 1 nM insulin for 18 h and then pelleted at 500 × g for 5 min. The cell pellet was lysed in 100 µl of ice-cold 1 mM phenylmethylsulfonyl fluoride and 50 mM Tris, pH 7.4, by 10 passages through a 25-gauge needle. Lysates were centrifuged at 16,000 × g for 10 min, and the supernatant fraction was adjusted to 0.4 mM EGTA, 0.4 mM NaPO4, 0.9 mM phenylmethylsulfonyl fluoride, 1 µM [gamma -32P]ATP (100 µCi/nmol), 45 mM Tris, and 20 mM HEPES, pH 7.1, at 22 °C with or without 10 mM MgCl2 as a cofactor. This kinase mixture was then added to affinity-purified GST-IRS-1-(511-772) (bound to glutathione-Sepharose), and the reaction was allowed to proceed for 15 min at 22 °C. Reactions were terminated by addition of phosphate-buffered saline with 1 mM EDTA and were linear with respect to time and amount of kinase. Phosphorylated IRS-1-(511-772) was then removed from the solid phase with Precision protease as described above. For JAK1 kinase assays, IRS-1-(511-772) was prephosphorylated in the absence of [gamma -32P]ATP and then used in JAK1 kinase assays as described above at a concentration of 5 µg/ml.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

OA Blocks IFN-alpha -dependent IRS-1/PI3K Activation and Association-- Serine phosphorylation of IRS-1 induced by OA treatment of 3T3-L1 adipocytes stops insulin-dependent activation of PI3K (26). To determine if IFN-alpha -mediated PI3K activation was prevented by OA, IRS-1-associated PI3K activity was examined in U266 cells pretreated with 1 µM OA for 30 min. Fig. 1A demonstrates that 1000 units/ml IFN-alpha induced 20-, 22-, and 10-fold increases in IRS-1-associated PI3K activity at 5, 10, and 30 min, respectively, and that pretreatment of cells with OA blocked this response. OA did not inhibit PI3K activity directly because PI3K activity in PI3K p85 immune complexes from OA-treated cells was no different than that from non-OA-treated cells (data not shown). To determine if this failure to activate PI3K was due to a loss of IFN-alpha -dependent IRS-1/PI3K association, Western analysis was performed. Fig. 1B shows that IFN-alpha -dependent IRS-1/PI3K association was increased at 5, 10, and 30 min (as measured by Western detection of PI3K p85) and that OA inhibited this association. To determine if this OA-dependent decline in IRS-1/PI3K association was due to an inhibition of IFN-alpha -dependent tyrosine phosphorylation of IRS-1, Western analysis was again performed. Fig. 1C shows that IFN-alpha increased IRS-1 tyrosine phosphorylation at 5, 10, and 30 min and that OA blocked detectable tyrosine phosphorylation of IRS-1. To confirm that OA did not measurably alter IRS-1, p85, and JAK1 protein levels and the ability of these proteins to be immunoprecipitated by their respective antibodies, Western analysis was performed. Fig. 1D demonstrates that IRS-1, p85, and JAK1 protein levels and their ability to be immunoprecipitated were unaffected by OA. Finally, to show that OA did not affect JAK1 autophosphorylation or its ability to phosphorylate in vitro substrates, JAK1 kinase assays were performed. Fig. 1E demonstrates that JAK1 isolated from OA-treated cells phosphorylated recombinant IRS-1-(511-772) as well as JAK1 recovered from non-OA-treated cells and that JAK1 autophosphorylation was unchanged. Taken together, these findings indicate that OA blocks IFN-alpha -dependent IRS-1/PI3K association by a mechanism that inhibits tyrosine phosphorylation of IRS-1, but does not alter JAK1 kinase activity.


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  		Fig. 1.   OA blocks IFN-alpha -dependent IRS-1/PI3K activation and association. A, U266 cells were pretreated with (OA) or without (Control) 1 µM OA for 30 min and then treated with 1000 units/ml IFN-alpha for the times indicated. PI3K (PIP) activity was measured in IRS-1 immunoprecipitates. B, cells were treated as described for A, and Western analysis was used to detect PI3K p85 (p85) in IRS-1 immunoprecipitates (IP) using an anti-p85 antibody. C, cells were treated as described for A, and Western analysis was used to detect IRS-1 tyrosine phosphorylation using an anti-phosphotyrosine antibody (pY). D, cells were pretreated with (+) or without (-) 1 µM OA for 30 min, and Western analysis was performed on IRS-1, p85, and JAK1 immunoprecipitates using the antibodies indicated. E, cells were pretreated with (+) or without (-) OA for 30 min as indicated and then treated with or without 1000 units/ml IFN-alpha for 5 min. JAK1 was immunoprecipitated, and JAK1 kinase assays were performed in the presence (right panel) or absence (left panel) of IRS-1-(511-772). All data are representative of triplicate experiments.

JAK1-dependent Phosphorylation of IRS-1 Is Inhibited by OA-- Serine phosphorylation of IRS-1 blocks insulin receptor-dependent tyrosine phosphorylation of IRS-1 (26-35). To determine if OA inhibited JAK1-dependent IRS-1 tyrosine phosphorylation, JAK1 kinase assays were performed. Fig. 2A demonstrates that when IRS-1 isolated from OA-treated cells was used as a substrate for JAK1, IFN-alpha -dependent JAK1 phosphorylation was not observed. In contrast, when IRS-1 from non-OA-treated cells was used as a substrate for JAK1, IFN-alpha induced a 5-fold increase in JAK1-dependent IRS-1 phosphorylation. To examine the phosphorylation state of IRS-1 isolated from OA-treated cells, phosphoamino acid analysis was performed. These experiments showed that IRS-1 was predominantly phosphorylated on serine residues and that no tyrosine phosphorylation was detected (Fig. 2B). Taken together, these findings indicate that serine phosphorylation of IRS-1 induced by OA renders IRS-1 a poorer substrate for JAK1.


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  		Fig. 2.   JAK1-dependent phosphorylation of IRS-1 is inhibited by OA. A, U266 cells were treated with (+) or without (-) 1 µM OA for 30 min as indicated, and IRS-1 was isolated by immunoprecipitation with an anti-IRS-1 antibody. IRS-1 was eluted from the antibody and used as a substrate in JAK1 kinase assays in which JAK1 was isolated from U266 cells treated for 5 min with or without 1000 units/ml IFN-alpha as indicated. B, phosphorylated IRS-1 was isolated from U266 cells metabolically labeled with 32Pi and treated with (+) or without (-) 1 µM OA for 30 min. Phosphoamino acid analysis was then performed on the recovered IRS-1. pSer, phosphoserine; pThr, phosphothreonine; pTyr, phosphotyrosine. All data are representative of triplicate experiments.

Chronic Insulin Treatment Inhibits IFN-alpha -dependent IRS-1/PI3K Activation and Association-- Chronic hyperinsulinemia induces serine phosphorylation of IRS-1 and reduces insulin signaling (33, 35, 40-42). To determine if chronic insulin treatment inhibited IFN-alpha -dependent activation of PI3K, IRS-1-associated PI3K activity was examined in U266 cells pretreated with 1 nM insulin for 18 h. Fig. 3A demonstrates that 1000 units/ml IFN-alpha induced a 10-fold increase in IRS-1-associated PI3K activity at 5 min and that pretreatment of cells with insulin reduced this response by 50%. Chronic insulin treatment did not inhibit PI3K activity directly because PI3K activity in PI3K p85 immune complexes from insulin-treated cells was no different than that from non-insulin-treated cells (data not shown). To determine if this reduction in PI3K activation was due to a loss of IFN-alpha -dependent IRS-1/PI3K association, Western analysis was performed. Fig. 3B shows that IFN-alpha increased IRS-1/PI3K association at 5 min (as measured by detection of PI3K p85) and that chronic insulin treatment reduced this association by 50%. To further examine the impact of chronic insulin treatment on IRS-1/PI3K association, IRS-1 present in PI3K immune complexes was examined by Western analysis (Fig. 3C). As in Fig. 3B, chronic insulin treatment reduced IFN-alpha -dependent IRS-1/PI3K association, and comparable low amounts of IRS-1 were associated with PI3K before and after chronic insulin treatment in cells not treated with IFN-alpha . To determine if this insulin-dependent decline in IRS-1/PI3K association after IFN-alpha treatment was due to an inhibition of IFN-alpha -dependent tyrosine phosphorylation of IRS-1, Western analysis was again performed. Fig. 3D shows that IFN-alpha increased IRS-1 tyrosine phosphorylation at 5 min and that chronic insulin treatment reduced IFN-alpha -dependent tyrosine phosphorylation of IRS-1 by 50%. Additionally, chronic insulin treatment did not alter JAK1 activity in that IFN-alpha -activated JAK1 isolated from chronically insulin-treated cells phosphorylated recombinant IRS-1-(511-772) as well as JAK1 recovered from non-insulin-treated cells (data not shown). Finally, to confirm that chronic insulin treatment did not measurably alter IRS-1, p85, and JAK1 protein levels and the ability of these proteins to be immunoprecipitated by their respective antibodies, Western analysis was performed. Fig. 3E demonstrates that IRS-1, p85, and JAK1 protein levels and the ability to be immunoprecipitated were unaffected by chronic insulin treatment. Taken together, these findings indicate that chronic insulin treatment inhibits IFN-alpha -dependent IRS-1/PI3K association by a mechanism that reduces tyrosine phosphorylation of IRS-1, but does not alter JAK1 kinase activity.


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  		Fig. 3.   Chronic insulin treatment inhibits IFN-alpha -dependent IRS-1/PI3K activation and association. A, U266 cells were pretreated with (+) or without (-) 1 nM insulin for 18 h as indicated and then treated with (closed bars) or without (open bars) 1000 units/ml IFN-alpha for 5 min. PI3K activity was measured in IRS-1 immunoprecipitates. Data are representative of triplicate experiments ± S.E. B, cells were treated as described for A, and Western analysis was used to detect PI3K p85 (p85) in IRS-1 immunoprecipitates (IP) using an anti-p85 antibody. C, cells were treated as described for A, and Western analysis was used to detect IRS-1 in PI3K p85 (p85) immunoprecipitates using an anti-IRS-1 antibody. D, cells were treated as described for A, and Western analysis was used to detect IRS-1 tyrosine phosphorylation using an anti-phosphotyrosine antibody (pY). E, cells were treated with (+) or without (-) 1 nM insulin for 18 h as indicated, and Western analysis was performed on IRS-1, p85, and JAK1 immunoprecipitates using the antibodies indicated. Data in B-E are representative of triplicate experiments.

Serine Phosphorylation of IRS-1-(511-772) Inhibits Its Phosphorylation by JAK1-- Phosphorylation of IRS-1 by serine kinases renders it a poorer substrate for the insulin receptor (27-35). To determine if serine phosphorylation of IRS-1 inhibits its ability to act as a JAK1 substrate, kinase assays were performed. Fig. 4A shows that plasma membrane-depleted lysates from U266 cells treated with 1 nM insulin for 18 h contained kinase activity that phosphorylated IRS-1-(511-772) exclusively on serine residues. Fig. 4B demonstrates that phosphorylation of IRS-1-(511-772) by plasma membrane-depleted serine kinase activity reduced by 50% the ability of IRS-1-(511-772) to serve as a substrate for IFN-alpha -activated JAK1. Fig. 4C demonstrates that IRS-1-(511-772) was a substrate for the serine kinase PAS kinase (34) and that phosphorylation of IRS-1-(511-772) by PAS kinase reduced by 75% the ability of IRS-1-(511-772) to serve as a substrate for IFN-alpha -activated JAK1 (Fig. 4D). These results indicate that serine phosphorylation of IRS-1 inhibits its ability to act as a JAK1 substrate.


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  		Fig. 4.   Serine phosphorylation of IRS-1-(511-772) inhibits its phosphorylation by JAK1. A, Plasma-membrane depleted cell lysates were prepared from U266 cells treated with 1 nM insulin for 18 h. Kinase assays using these lysates were performed with IRS-1-(511-772) as a substrate in the presence (+) or absence (-) of 10 mM MgCl2 (left panel). Phosphoamino acid analysis was performed on IRS-1-(511-772) from the + lanes (right panel). B, IRS-1-(511-772) was prephosphorylated with (Insulin +) or without (Insulin -) serine kinase activity generated in A and then used as a substrate for JAK1 isolated from U266 cells treated with (+) or without (-) 1000 units/ml IFN-alpha for 5 min. C, PAS kinase was affinity-purified from U266 cells using GST-p85. PAS kinase assays were performed using IRS-1-(511-772) as a substrate in the presence (+) or absence (-) of 10 mM MgCl2. D, IRS-1-(511-772) was prephosphorylated as described for C without [gamma -32P]ATP and then used as a substrate for IFN-alpha -activated JAK1 isolated from U266 cells treated with 1000 units/ml IFN-alpha for 5 min. All data are representative of triplicate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These data establish that serine phosphorylation of IRS-1 renders it a poorer substrate for JAK1. Western analysis and PI3K assays demonstrated that IFN-alpha -dependent IRS-1 tyrosine phosphorylation and IRS-1/PI3K association and activity were blocked by OA treatment and that this was not due to an effect of OA on JAK1 (Fig. 1, A-D). Isolation of IRS-1 from OA-treated cells showed that OA-dependent serine phosphorylation of IRS-1 completely inhibited the ability of IFN-alpha -activated JAK1 to phosphorylate IRS-1 (Fig. 2). Likewise, chronic insulin stimulation reduced by 50% the ability of IFN-alpha to stimulate IRS-1 tyrosine phosphorylation and IRS-1/PI3K association and activity (Fig. 3). More importantly, serine phosphorylation of IRS-1-(511-772) by serine kinases derived from chronically insulin-stimulated cells and by PAS kinase reduced by 50 and 75%, respectively, the ability of IFN-alpha -activated JAK1 to phosphorylate IRS-1-(511-772) (Fig. 4). Taken together, these findings indicate that serine phosphorylation of IRS-1 reduces the ability of IRS-1 to serve as a JAK1 substrate, that IRS-1 serine phosphorylation inhibits signal transduction in pathways outside the insulin system, and that hyperinsulinemia may alter signaling of JAK1-dependent cytokine receptors.

Inhibition of PP1 and PP2A serine phosphatases by OA and calyculin A increase IRS-1 serine phosphorylation and leads to decreased insulin receptor-mediated IRS-1 tyrosine phosphorylation (26, 33). Chronic insulin treatment has also been shown to induce serine phosphorylation of IRS-1 and to inhibit insulin receptor-dependent phosphorylation of IRS-1 (33, 35, 40-42). Recently, the region of IRS-1 susceptible to chronic insulin treatment-dependent serine phosphorylation has been reported, and it appears to reside between amino acids 530 and 843 (35). The kinase responsible for this phosphorylation is unknown, but appears to be insensitive to inhibitors of protein kinases C and A, PI3K, and mitogen-activated protein kinase (35). We have identified a kinase (PAS kinase) that can serine phosphorylate IRS-1 and inhibit the ability of IRS-1 to serve as an insulin receptor substrate (34, 37). This kinase associates with the p85 subunit of PI3K through SH2 domain interactions and phosphorylates IRS-1 in IRS-1/PI3K complexes after insulin stimulation (37). Here, we show that PAS kinase can phosphorylate IRS-1-(511-772) and that this phosphorylation inhibits the ability of IFN-alpha -activated JAK1 to subsequently phosphorylate IRS-1-(511-772).

Although serine phosphorylation of IRS-1 decreases the ability of the insulin receptor and now JAK1 to phosphorylate IRS-1, the mechanism of this effect is not clearly delineated. In the insulin signaling system, serine phosphorylation of IRS-1 within the IH1 phosphotyrosine-binding domain appears to impair NPXY-mediated IRS-1/insulin receptor association (33), thus abrogating direct IRS-1/insulin receptor interaction. Like the insulin receptor, the IL-4 receptor contains an NPXY motif, and this motif appears to coordinate the formation of receptor/JAK/IRS-1 complexes, which result in IRS-1 tyrosine phosphorylation (43). The IFN-alpha receptor does not contain an NPXY motif and may rely on the IRS-1 IH1 pleckstrin homology domain to coordinate receptor/JAK/IRS-1 association and subsequent IRS-1 phosphorylation (8). This suggests that serine phosphorylation within the IRS-1 IH1 pleckstrin homology domain might be important for preventing IFN-alpha -activated JAK1-dependent tyrosine phosphorylation of IRS-1. We show here, however, that IFN-alpha -activated JAK1 can phosphorylate IRS-1-(511-772) and that serine phosphorylation of IRS-1-(511-772) inhibits this effect. This is important in that IRS-1-(511-772) does not contain either the IRS-1 IH1 pleckstrin homology or IH2 phosphotyrosine-binding domain and suggests that other regions of IRS-1 may be important in IRS-1/JAK1 interactions.

Hyperinsulinemia and insulin resistance are prominent features in both syndrome X and the development of type 2 diabetes mellitus (44). However, the pathogenesis of the multiple complications and conditions associated with these diseases is not yet understood (45). We show here that chronic insulin treatment and IRS-1 serine phosphorylation decrease JAK1-mediated IRS-1 tyrosine phosphorylation and IRS-1/PI3K association, suggesting that cytokine signal transduction may be altered during hyperinsulinemia. Currently, a rapidly growing number of hormone and cytokine receptors appear to signal through JAK and IRS family members, and this appears to be critical to hormone/cytokine function (1). This is most clearly understood in IL-4 signaling, where IRS function has been shown to be critical to IL-4-dependent mitogenesis and anti-apoptosis (4, 46). Additionally, site-specific mutagenesis of the phosphotyrosine-binding domain-binding motif in the IL-4 receptor reduces both IRS and STAT6 tyrosine phosphorylation and abolishes the effect of IL-4 on the induction of DNA binding activity and CD23 induction (47). Thus, by inducing IRS serine phosphorylation, hyperinsulinemia may potentially contribute to the pathogenesis of syndrome X/type 2 diabetes mellitus complications by disrupting JAK-mediated cytokine and hormone signaling pathways that use IRS. In summary, we show that OA and chronic insulin treatment inhibit IFN-alpha -dependent IRS-1 tyrosine phosphorylation and IRS-1/PI3K association and activity. More importantly, we show that these effects are mediated by serine phosphorylation of IRS-1. We conclude that IRS-1 serine phosphorylation plays an inhibitory role in signaling pathways outside the insulin system and suggest that hyperinsulinemia may alter signaling of JAK1-dependent cytokine receptors through serine phosphorylation of IRS-1.

    FOOTNOTES

* This work was supported by Grant CA-61931 from the National Institutes of Health; by Grant 97-GB-02 from the American Heart Association, Illinois Affiliate; and by grants from the Macula Foundation and the American Diabetes Association (all to G. G. F.).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.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Pathology, College of Medicine, 506 South Mathews Ave., University of Illinois at Urbana-Champaign, Urbana, IL 61801. Tel.: 217-244-8839; Fax: 217-244-5617; E-mail: freun@ux1.cso.uiuc.edu.

    ABBREVIATIONS

The abbreviations used are: IRS, insulin receptor substrate; IL, interleukin; IFN, interferon; PI3K, phosphatidylinositol 3'-kinase; OA, okadaic acid; PAS kinase, PI3K-associated serine kinase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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