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Recipient of a Career Development Award from the American Cancer Society. To whom correspondence and reprint requests should be addressed: Division of Hematology-Oncology, Loyola University of Chicago, Bldg. 112, 2160 South First Ave., Maywood, IL 60153 . Tel.: 708-327-3304; Fax: 708-216-2319
Hines Veterans Administration Medical Center, Hines, Illinois 60141
∗ This work was supported by grants from the Department of Veterans Affairs and the Hairy Cell Leukemia Foundation (to L. C. P.), and by National Institutes of Health Grants DK43808 and DK38712 (to M. F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Interferon-α (IFNα) induces rapid tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), a docking protein with multiple tyrosine phosphorylation sites that bind to the Src homology 2 (SH2) domains of various signaling proteins. During IFNα stimulation, the p85 regulatory subunit of the phosphatidylinositol 3′-kinase binds via its SH2 domains to tyrosine-phosphorylated IRS-1, and phosphatidylinositol 3′-kinase activity is detected in association with IRS-1. Thus, IFNα responses occur by activation of the IRS signaling system, which it shares with insulin, insulin-like growth factor-1, and interleukin-4.
), and their activation early in the IFNα signaling cascade is presumed to regulate tyrosine phosphorylation of various downstream signaling molecules. Expression of Jak-1 and Tyk-2 rescues an IFNα response in certain insensitive cell lines, suggesting that these tyrosine kinases or related members of the Janus family are essential for IFNα action(
Several proteins are substrates for IFNα-dependent tyrosine kinase activity. In response to IFNα treatment of cells, the Stat-2, Stat-1α, and Stat-1β components of the transcriptional activator ISGF3α are rapidly phosphorylated on tyrosine and associate with a 48-kDa protein (ISGF3γ) to form an active complex(
). This complex translocates to the nucleus and initiates gene transcription during binding to interferon-stimulated response elements (10-13). In addition, the vav proto-oncogene product (p95vav) is tyrosine-phosphorylated during IFNα stimulation (
); however, its precise role in the signal transduction of IFNα remains to be determined. The involvement of multiple pathways in IFNα signaling is consistent with its pleiotropic biological effects on cells and tissues.
Many growth factor receptors, including those for epidermal growth factor and platelet-derived growth factor, associate directly through their autophosphorylation sites with a common set of signaling proteins that contain SH2 domains, including the phosphatidylinositol (PI) 3′-kinase, Grb-2, SH-PTP2, phospholipase Cγ, and Ras-GAP(
). We report that IFNα also engages the IRS signaling system, as evidenced by the rapid IFNα-dependent tyrosine phosphorylation of IRS-1 in several hematopoietic cell lines. We also demonstrate that the p85 regulatory subunit of the PI 3′-kinase binds via its SH2 domains the IFNα-induced phosphorylated form of IRS-1, and that PI 3′-kinase activity can be detected in association with IRS-1.
Cells and Reagents
The U-266 (human myeloma), Daudi (lymphoblastoid), and MOLT-4 (acute T-cell leukemia) cell lines were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.) or 10% (v/v) defined calf serum (Hyclone Laboratories) and antibiotics. Human recombinant IFNα2 was provided by Hoffmann-La Roche and Schering-Plough. The antiphosphotyrosine monoclonal antibody (4G-10) and the monoclonal antibody against the regulatory subunit of PI 3′-kinase were obtained from Upstate Biotechnology (Lake Placid, NY) and were used for immunoblotting. Nonimmune rabbit IgG was obtained from Sigma. The polyclonal αIRS-1PH antibody was raised against a GST fusion protein of a region of rat IRS-1 (amino acid residues 1-135) containing its pleckstrin homology domain. An antibody (αIRS-1CT) against a synthetic peptide (TYASINFQKQPEDRQ), corresponding to a sequence present in the C terminus of IRS-1, was raised in rabbits. The polyclonal antibodies against recombinant rat IRS-1 and the p85 regulatory subunit of PI 3′-kinase have been described elsewhwere (
Cells were stimulated with the indicated amounts of IFNα or insulin for the indicated periods of time. After stimulation, the cells were rapidly centrifuged and lysed in a phosphorylation lysis buffer (0.5-1% Triton X-100, 150 mM NaCl, 200 μM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5 mM magnesium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin). Cell lysates were immunoprecipitated with the indicated antibodies and, after five washes with phosphorylation lysis buffer containing 0.1% Triton X-100, were analyzed by SDS-PAGE and transferred onto nitrocellulose (Schleicher and Schuell) or polyvinylidene difluoride (Immobilon) filters, and the residual binding sites on the filters were blocked by incubating with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20, 10% bovine serum albumin) for 1-3 h at room temperature or overnight at 4°C. The filters were subsequently incubated with the antiphosphotyrosine monoclonal antibody and developed using an enhanced chemiluminesence (ECL) kit following the manufacturer's procedure (Amersham).
Preparation of Glutathione S-Transferase Fusion Proteins
Glutathione S-transferase fusion proteins containing the nSH2 and cSH2 domains of PI 3′-kinase (
). Briefly, after 2 h of additional growth, bacteria were lysed by sonication in phosphate-buffered saline (PBS), pH 7.0. Lysed bacteria were spun for 25 min at 14,000 rpm at 4°C, and the supernatant was immobilized on glutathione-Sepharose beads (Pharmacia) and used for binding assays.
Assays for PI 3′-Kinase Activity
PI 3′-kinase activity assays on αIRS-1PH immunoprecipitates obtained from IFNα- or insulin-stimulated cells were performed as described previously(
). Briefly, after IFNα or insulin stimulation, serum-starved Daudi cells were washed once with ice-cold PBS and twice with 20 mM Tris, pH 7.5, containing 137 mM NaCl, 1 mM MgCl2, and 100 μM Na3VO4 (Buffer A). The cells were lysed in 1 ml of Buffer A containing 1% Nonidet P-40, 10% glycerol, and 0.35 mg/ml phenylmethylsulfonyl fluoride, and insoluble material was removed by centrifugation. Cell lysates were immunoprecipitated with the indicated antibodies, and immunoprecipitates were washed in PBS with 1% Nonidet P-40 and 100 μM Na3VO4 (three times), 100 mM Tris, pH 7.5, containing 500 mM LiCl2 and 100 μM Na3VO4 (three times), and 10 mM Tris, pH 7.5, containing 100 mM NaCl, 1 mM EDTA, and 100 μM Na3VO4 (twice). The pellets were resuspended in 50 μl of 10 mM Tris, pH 7.5, containing 100 mM NaCl and 1 mM EDTA. 10 μl of 100 mM MnCl2 and 10 μl of phosphatidylinositol (2 μg/μl) sonicated in 10 mM Tris, pH 7.5, 1 mM EGTA were added to each pellet. The reaction was started by the addition of 10 μl of 440 μM ATP containing 30 μCi of [32P]ATP. After incubation for 10 min at 22°C, the reaction was stopped by the addition of 20 μl of 8 N HCl and 160 μl of CHCl3:methanol (1:1). The samples were centrifuged, and the lower organic phase was removed and applied to a silica gel TLC plate which was coated with 1% potassium oxalate. TLC plates were developed in CHCl3:CH3OH:H2O:NH4OH (60:47:11.3:2), dried, and visualized and quantitated on a Molecular Dynamics PhosphorImager.
RESULTS AND DISCUSSION
We performed experiments in which the patterns of tyrosine phosphorylation induced by IFNα or insulin in hematopoietic cells were studied. We noticed that both IFNα and insulin stimulate tyrosine phosphorylation of a common 170-kDa protein in U-266 cells (Fig. 1). The molecular mass of this protein was similar to the mass of the insulin receptor substrate-1(
), suggesting that IFNα uses an IRS signaling protein to mediate certain biologic responses.
To determine if IRS-1 is involved in IFNα signaling, lysates from control or stimulated U-266 or Daudi cells were immunoprecipitated with polyclonal antibodies against IRS-1 and immunoblotted with an antiphosphotyrosine antibody. Basal tyrosine phosphorylation of a 170-kDa protein, corresponding to IRS-1, was consistently detected in U-266 but not in Daudi cells. However, IFNα and insulin strongly stimulated tyrosine phosphorylation of IRS-1 in both cell lines (Fig. 2, A and B, and data not shown). The IFNα-induced phosphorylation of IRS-1 was rapid and transient, occurring within 1 min of treatment of Daudi or MOLT-4 cells and diminishing after 90 min (Fig. 2C). It was also dose-dependent, exhibiting a similar response pattern with the phosphorylation of Stat proteins (Fig. 2D).
After tyrosine phosphorylation, IRS-1 binds to several SH2 proteins, including the 85-kDa regulatory subunit (p85) of the PI 3′-kinase(
). To determine whether IRS-1 binds p85 during IFNα stimulation, immunoprecipitates obtained with an anti-p85 antibody from Daudi cell lysates were immunoblotted with antiphosphotyrosine (4G-10). Following IFNα or insulin treatment of the cells, a 170-kDa tyrosine-phosphorylated protein was detected in the anti-p85 immunoprecipitates. This phosphoprotein co-migrated with IRS-1 immunoprecipitated directly with αIRS-1PH (Fig. 3A). Moreover, anti-IRS-1 immunoprecipitates contained p85α only after IFNα or insulin stimulation (Fig. 3B). The tyrosine-phosphorylated form of IRS-1 from IFNα- or insulin-stimulated cells also bound to a GST fusion protein containing the N-terminal SH2 domain of p85 (Fig. 3C and 3D). Similar results were obtained using a GST fusion protein containing the C-terminal SH2 domain of p85.2(
). To determine whether PI 3′-kinase activity is detected in association with IRS-1 during IFNα stimulation, PI 3′-kinase assays were carried out on αIRS-1 immunoprecipitates. Before stimulation, a basal level of PI 3′-kinase activity was detected in the αIRS-1 immunoprecipitates, which was equivalent to the nonspecific activity detected in immunoprecipitates with preimmune rabbit serum (Fig. 4). IFNα or insulin stimulated the association of PI 3′-kinase activity with IRS-1 that was immunoprecipitated specifically with αIRS-1PH (Fig. 4). It is likely that the PI 3′-kinase is activated by IFNα during its association with IRS-1.
Considerable progress has been made in our understanding of IFNα signaling between the plasma membrane and nucleus. The Jak-Stat pathway provides a plausible mechanism for the regulated assembly of ISGF-3, which regulates expression of genes containing the IFN-stimulated response elements(
). The molecular mechanism used by IFNα to regulate other signaling pathways, however, is poorly understood. Our finding that IFNα stimulates tyrosine phosphorylation of IRS-1 suggests the existence of a pathway for the regulated engagement of additional SH2 signaling proteins during IFNα stimulation. It will be important to identify other SH2 proteins engaged by IRS-1 in response to IFNα, as well as in response to IFNβ and IFNω, which also induce its phosphorylation on tyrosine residues.2
IRS-1 plays a central role in the signal transduction of insulin and interleukin-4, and its signaling functions appear to be essential for the mitogenic effects of these ligands(
). These tyrosine residues play a dual role as substrates for upstream tyrosine kinases and as specific docking sites for downstream SH2 proteins. At least eight tyrosine residues in IRS-1 undergo phosphorylation by the activated insulin receptor, including residues 460, 608, 628, 939, and 987, which are in YXXM/YMXM motifs and bind to p85 which activates the PI 3′-kinase(
). Studies to determine the motifs of IRS-1 that are tyrosine-phosphorylated in response to IFNα should provide valuable information on the mechanisms by which the signaling specificity is established through the common use of this protein by different cytokines and growth factors.
We currently think that the insulin receptor tyrosine kinase regulates the insulin-dependent phosphorylation of IRS proteins, whereas the receptors for IL-4 and IFNα use Janus family tyrosine kinases to accomplish such phosphorylation; however, direct evidence that IRS-1 acts as a substrate for Janus kinases during IFNα stimulation remains to be obtained. Many receptors activate the Janus family of tyrosine kinases, but most of them do not phosphorylate IRS-1. The identity of the elements responsible for this selectivity are unknown. Interestingly, the receptors for insulin, IGF-1, and IL-4 contain a common amino acid sequence motif, LxxxxNPxYxss, which appears to contribute to the interaction of these receptors with IRS-1(
) suggested that IL-4 attenuates the transcriptional activation of IFN-induced cellular gene expression in monocytes and related cell lines. Although the mechanism of this phenomenon is unknown, our results raise the possibility of an antagonism between IFNα and IL-4 occurring through the common use of proteins in the IRS signaling system. If this hypothesis is correct, then insulin and IGF-1 may also influence IFNα signaling. Additional work in this area should reveal important relationships between these apparently distinct factors and others as well.
We thank Drs. Michael Brunda (Hoffman-La Roche) and Paul Trotta (Schering-Plough) for providing us with IFNα.