Analysis of interleukin-2-dependent signal transduction through the Shc/Grb2 adapter pathway. Interleukin-2-dependent mitogenesis does not require Shc phosphorylation or receptor association.

The interleukin (IL)-2 receptor system has previously been shown to signal through the association and tyrosine phosphorylation of Shc. This study demonstrates that the IL-2 receptor β (IL-2Rβ) chain is the critical receptor component required to mediate this effect. The use of IL-2Rβ chain deletion mutants transfected into a Ba/F3 murine cell model describes a requirement for the IL-2Rβ “acid-rich” domain between amino acids 315 and 384 for Shc tyrosine phosphorylation and receptor association. COS cell co-transfection studies of IL-2Rβ chain constructs containing point mutations of tyrosine to phenylalanine along with the tyrosine kinase Jak-1 and a hemagglutinin-tagged Shc revealed that the motif surrounding phosphorylated tyrosine 338 within the acid-rich domain of the IL-2Rβ is a binding site for Shc. Deletion of this domain has previously been shown to abrogate the ability of IL-2 to activate Ras but does not affect IL-2-dependent mitogenesis in the presence of serum. Proliferation assays of Ba/F3 cells containing IL-2Rβ chain deletion mutants in serum-free medium with or without insulin shows that deletion of the acid-rich domain does not affect IL-2-driven mitogenesis regardless of the culture conditions. This study thus defines the critical domain within the IL-2Rβ chain required to mediate Shc binding and Shc tyrosine phosphorylation and further shows that Shc binding and phosphorylation are not required for IL-2-dependent mitogenesis. Neither serum nor insulin is required to supplement the loss of induction of the Shc adapter or Ras pathways, which therefore suggests a novel mechanism for mitogenic signal transduction mediated by this hematopoietin receptor.

The interleukin (IL)-2 receptor system has previously been shown to signal through the association and tyrosine phosphorylation of Shc. This study demonstrates that the IL-2 receptor ␤ (IL-2R␤) chain is the critical receptor component required to mediate this effect. The use of IL-2R␤ chain deletion mutants transfected into a Ba/F3 murine cell model describes a requirement for the IL-2R␤ "acid-rich" domain between amino acids 315 and 384 for Shc tyrosine phosphorylation and receptor association. COS cell co-transfection studies of IL-2R␤ chain constructs containing point mutations of tyrosine to phenylalanine along with the tyrosine kinase Jak-1 and a hemagglutinin-tagged Shc revealed that the motif surrounding phosphorylated tyrosine 338 within the acidrich domain of the IL-2R␤ is a binding site for Shc. Deletion of this domain has previously been shown to abrogate the ability of IL-2 to activate Ras but does not affect IL-2-dependent mitogenesis in the presence of serum. Proliferation assays of Ba/F3 cells containing IL-2R␤ chain deletion mutants in serum-free medium with or without insulin shows that deletion of the acid-rich domain does not affect IL-2-driven mitogenesis regardless of the culture conditions. This study thus defines the critical domain within the IL-2R␤ chain required to mediate Shc binding and Shc tyrosine phosphorylation and further shows that Shc binding and phosphorylation are not required for IL-2-dependent mitogenesis. Neither serum nor insulin is required to supplement the loss of induction of the Shc adapter or Ras pathways, which therefore suggests a novel mechanism for mitogenic signal transduction mediated by this hematopoietin receptor.
Interleukin-2 (IL-2) 1 is a multifunctional cytokine that has been shown to affect the physiology of cells of immune and nonimmune tissue (reviewed in Ref. 1) by direct interaction with a high or intermediate affinity IL-2 receptor (IL-2R). The high affinity IL-2R (K d ϭ 10 Ϫ11 ) is composed of three receptor subunits (reviewed in Ref. 2); ␣ (55 kDa), ␤ (75 kDa), and the common ␥ chain (␥ c , 64 kDa), which is shared by several hematopoietic cytokine receptors (3). The intermediate affinity receptor (K d ϭ 10 Ϫ9 ) is a ␤-␥ c complex only (2). Signal transduction by IL-2 minimally requires the intermediate affinity ␤-␥ c receptor complex and does not require the ␣ chain (2). Thus the apparent function of the ␣ chain is to affect IL-2 affinity only and not the mechanism of IL-2 signal transduction.
Signal transduction by IL-2 is initiated by the activation of several tyrosine kinases associated with specific receptor molecules of the IL-2R. Among these are members of the Src family of tyrosine kinases, which have been shown to functionally couple to the IL-2R␤ chain (4 -6); the Janus family tyrosine kinase Jak-1, which associates with the IL-2R␤ chain (7,8); and Jak-3 (9 -11), which associates with the ␥ c chain (7,8). The first detectable event following treatment of T cells with IL-2 is the formation of an activated ␣-␤-␥ c IL-2R complex containing tyrosine-phosphorylated and activated Jak-1 and Jak-3 (7,8,10). This is soon followed by the activation of Src family tyrosine kinases (4 -6) and the tyrosine phosphorylation of multiple substrates.
Among the proteins that are tyrosine phosphorylated in response to IL-2 is the recently identified protein Shc (12). Shc is an SH2 domain containing protein found as two dominant, widely expressed, and tyrosine-phosphorylated forms of 52 and 46 kDa as well as a 66-kDa protein with more restricted expression (13). Shc functions to link receptor tyrosine kinase activation and tyrosine phosphorylation to the downstream activation of Ras and Ras-like pathways (reviewed in Ref. 14) and accomplishes this by recognizing and binding to a phosphotyrosine-containing motif within the receptor or a receptorassociated protein. Binding to the receptor complex brings Shc into proximity for phosphorylation by an activated tyrosine kinase and establishes a secondary "docking" site for the protein Grb2 (15). The subsequent binding of Grb2 to the receptor complex brings guanine nucleotide releasing factor activity, such as Sos (16) or Vav (17), to the membrane where it can catalyze the release of GDP from inactive Ras-GDP, allowing the formation of an activated Ras-GTP complex. Thus it is clear that the activation of Ras via the Shc/Grb2 adapter pathway is dependent on the ability of Shc to recognize and bind to a tyrosine-phosphorylated receptor complex.
Tyrosine phosphorylation of p52Shc and activation of the Shc/Grb2 adapter pathway can be induced by a number of mitogenic cytokines and growth factors (13, 18 -22) and involves the ligand-dependent association of Shc with the receptor complex (23,24). As in other systems, Shc has been found associated with the IL-2R complex (25) and the induction of this pathway by IL-2 has been reported to correlate with the ability of IL-2 to elicit a mitogenic response (26). Because of this and the multi-protein nature of the IL-2R complex, we were interested in determining the precise mechanism by which IL-2 stimulates Shc/receptor binding and tyrosine phosphorylation.

MATERIALS AND METHODS
Tissue Culture-Human T cells were obtained from normal donors and isolated by counter flow centrifugal elutriation (27). Isolated T cells were activated and cultured for 3 days in RPMI media (Mediatech) supplemented with 10% fetal calf serum (Intergen), 1 g/ml phytohemagglutinin, glutamine, and antibiotics (28). Cells were recovered and G 1 -enriched (28) by washing several times in low pH RPMI media and culturing for 24 h in RPMI media supplemented with 1% fetal calf serum, glutamine, and antibiotics. Cells were treated with 100 nM recombinant human IL-2 or IL-4 (Peprotech) for up to 30 min as described (29), recovered by centrifugation, and prepared for lysis and immune precipitation.
Ba/F3 cells transfected with various IL-2R␤ chain deletion mutants (FL, AD, BD, SD, and BS) were maintained at 37°C in a humidified CO 2 incubator in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% WEHI-3B culture supernatant (30), glutamine, antibiotics, and 250 g/ml hygromycin-B (Calbiochem). IL-2 treatments were performed on these cells using 100 nM recombinant IL-2 at a cell density of 50 ϫ 10 6 cells/ml in RPMI 1640 medium supplemented with 5% fetal calf serum. Cells were incubated at 37°C for 15 min, recovered by centrifugation, and prepared for lysis and immune precipitation.
Transfections-Constructs containing IL-2R␤ chain deletion mutations were prepared as described (31), transfected into Ba/F3 cells, and cultured as described (31). Plasmid constructs containing wild type or mutant IL-2R␤ chain molecules with substitutions of phenylalanine for tyrosine were generated as described (32). The Jak-1 expression plasmid was prepared by subcloning the 4.2 kB murine Jak-1 cDNA (provided by O. Silvennoinen and J. Ihle) into the SalI/SmaI sites of pCMV4⌬ (provided by M. Feinberg). A hemagglutinin (HA) epitopetagged derivative of human Shc (containing two tandem copies of the sequence YPYDVPDYD at the 5Ј end) was prepared by polymerase chain reaction, and the 1.6-kilobase pair cDNA was subcloned into the HindIII/KpnI sites of the expression plasmid pCMV4Neo (provided by M. Feinberg). Plasmids were transfected into COS-7 cells (ATCC) using Lipofectamine (Life Technologies, Inc.) as described (33).
Anti-human Jak-3 and anti-Shc immune precipitation of human T cell lysates were performed on a 100 ϫ 10 6 cell lysate equivalent using 2 l of anti-Jak-3 antibody (9) or 1 l of anti-Shc antibody (Upstate Biotechnology Inc.). Anti-Shc immune precipitation of Ba/F3 cells was performed using a 300 ϫ 10 6 cell lysate equivalent and 4 l of anti-Shc antibody/sample. All immune precipitations were performed for 4 h to overnight at 4°C in the presence of 20 l of protein A-conjugated Sepharose (Sigma). Immune precipitations were washed 6 times in lysis buffer, and proteins were eluted by boiling in 50 l of 2 ϫ SDSpolyacrylamide gel electrophoresis sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon polyvinylidene difluoride membranes (Millipore), and probed using anti-phosphotyrosine immunoblotting.
IL-2R␤ immune precipitation was performed using anti-IL-2R␤ monoclonal antibody 561 (provided by R. Robb) directly conjugated to protein A-Sepharose (PAS). Preparation of 561-PAS was initiated by incubating 1 mg of 561 with 1 ml of packed PAS beads in 3 ml of lysis buffer overnight at 4°C. The beads were washed extensively with lysis buffer, 2 times with 0.1 M borate, pH 9.0 (BB) and resuspended in 10 mls of BB. Dimethyl pimelimidate (Sigma) was added to 20 mM, and beads were incubated at room temperature for 1 h. 561-PAS was washed once with BB, resuspended in 40 mM ethanolamine pH 8.0, and incubated for 1 h at room temperature. Beads were washed several times with alternating cycles of BB and 0.1 M glycine, pH 3.0. 561-PAS was resuspended at a final concentration of 1 mg/ml 561/packed beads in phosphatebuffered saline, supplemented with 0.1% sodium azide, and stored at 4°C.
For IL-2R␤ chain immune precipitations, treated Ba/F3 cells containing mutant IL-2R␤ chains or transfected COS-7 cells were lysed, clarified as described, and immune precipitated using 30 l of 561-PAS (30 g of antibody equivalent). Immune precipitations were incubated for 4 h at 4°C and washed six times with lysis buffer. Proteins were eluted by boiling in 50 l of nonreducing 2 ϫ SDS-polyacrylamide gel electrophoresis sample buffer (no ␤-mercaptoethanol). Samples were centrifuged, and the supernatant was removed and adjusted to 5% ␤-mercaptoethanol. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon polyvinylidene difluoride membranes (Millipore), and probed for Shc or Shc-HA by anti-Shc or anti-HA immunoblotting.
Immunoblotting-All immunoblotting was performed as described (34) using the following antibodies. For the detection of changes in tyrosine phosphorylation, immune precipitated samples on polyvinylidene difluoride paper were probed using 1 g/ml anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology Inc.). Anti-Shc immunoblotting was performed using 1 g/ml polyclonal anti-Shc antibody (Upstate Biotechnology Inc.). Anti-HA immunoblotting for the detection of hemagglutinin-tagged Shc was performed using polyclonal anti-HA tag antibody (BABCO). Anti-IL2R␤ chain immunoblotting was performed using 1 g/ml monoclonal antibody 561. All immunoblots were developed using the ECL developement kit following the manufacturer's direction (Amersham Corp.) Proliferation Assays-Ba/F3 cells containing various IL-2R␤ chain deletion mutants were cultured as described above and obtained 2 days after feeding. Cells were washed two times in QBSF-56 medium (Quality Biologicals Inc.), plated in 96-well culture plates at 5000 cells/well in QBSF-56 medium with or without insulin (Quality Biologicals Inc.), and supplemented with glutamine and antibiotics. Cells were treated with 50 ng/ml recombinant human IL-2 and cultured for 24 h at 37°C in a humidified CO 2 incubator. Cells were then pulsed with [ 3 H]thymidine for 6 h, and proliferation was quantitated as described (35).

RESULTS
The IL-2R␤ Chain Mediates IL-2-dependent Shc Phosphorylation-Several hematopoietic cytokines induce p52Shc tyrosine phosphorylation (13,19,21,22), which correlates with their ability to activate Ras and induce mitogenesis (26). IL-2 has been shown to induce Shc association with the IL-2 receptor complex (25), stimulate the tyrosine phosphorylation of Shc (12), and activate Ras in human T cells (36). For these reasons we were interested in determining the mechanism by which IL-2 induces Shc phosphorylation leading to activation of the Shc adapter pathway.
The IL-2 receptor complex contains four dominant tyrosinephosphorylated proteins: the IL-2R␤ chain, the common ␥ chain, and the receptor-associated tyrosine kinases Jak-1 and Jak-3. To determine which of these phosphotyrosine containing proteins was most important in mediating Shc phosphorylation we used the IL-4 receptor system, which also contains the common ␥ chain and activates Jak-1 and Jak-3 (7), and asked whether IL-4 was capable of inducing Shc phosphorylation in a manner similar to that observed with IL-2.
Activated human T cells were treated with IL-2 or IL-4 and assayed for receptor tyrosine kinase activation, as represented by increased tyrosine phosphorylation of Jak-3, and factor-dependent tyrosine phosphorylation of Shc. This analysis clearly demonstrates that IL-4 does not induce Shc tyrosine phosphorylation in spite of inducing receptor tyrosine kinase activity and phosphorylation of Jak-3 in human T cells (Fig. 1). This observation is supported by additional results that describe an inability of IL-4 to induce Shc tyrosine phosphorylation in other cell types (37). This therefore implies a direct role for the IL-2R␤ chain in controlling IL-2-dependent Shc phosphorylation.
The "Acid-rich" Domain of the IL-2R␤ Chain Is Required for Shc Tyrosine Phosphorylation and Shc Association with the IL-2R-Previously generated and characterized IL-2R␤ molecules containing various deletion mutations within the cytoplasmic domain (31) (Fig. 2) were transfected into murine IL-3-dependent Ba/F3 cells and analyzed for their ability to support IL-2-induced receptor tyrosine kinase activation and substrate phosphorylation leading to mitogenesis (summarized in Table I). As previously reported, only wild type (FL), acidrich domain deletion mutants (AD), and truncation deletion mutants from amino acid 384 to the carboxyl terminus (BD) were capable of supporting IL-2-induced tyrosine phosphorylation and mitogenesis (Table I and Ref. 31). We next asked whether there was a difference in the ability of these mutants to transduce IL-2-dependent signal leading to Shc phosphorylation. Receptor-containing Ba/F3 cells were treated with IL-2 and assayed for IL-2-induced Shc phosphorylation by anti-Shc immune precipitation followed by anti-phosphotyrosine immunoblotting. As shown in Fig. 3, the wild type receptor (FL) and the BD mutant, containing an intact acid-rich domain, were capable of inducing IL-2-dependent tyrosine phosphorylation of Shc. Deletion of this domain in the AD receptor resulted in an inability to transduce signal leading to Shc phosphorylation (Fig. 3). As a control, IL-2-induced tyrosine phosphorylation of murine Jak-3 and downstream substrates was verified in all three mutant receptor-containing cells (Ref. 31 and data not shown). SD-and BS-containing cells were incapable of inducing tyrosine phosphorylation of Shc or Jak-3 (data not shown), supporting previous observations (31). This analysis suggests that a domain within the acid-rich region of the IL-2R␤ is required to mediate IL-2-dependent Shc phosphorylation and may represent a binding site for Shc.
FL-, AD-, and BD-containing cells were treated with IL-2, the IL-2R␤ chain was immune precipitated, and an immunoblot was performed using an anti-Shc antibody. Results from this experiment show an IL-2-dependent association of Shc with FL and BD receptors but not in cells containing the AD mutant (Fig. 4). This clearly shows that IL-2-dependent tyrosine phosphorylation of Shc involves direct binding of Shc to a motif within the acid-rich domain of the receptor and implies a requirement for IL-2-dependent tyrosine phosphorylation of this region as a prerequisite for Shc association.
The Motif Surrounding Phosphorylated Tyrosine 338 within the IL-2R␤ Chain Cytoplasmic Domain Provides the Binding Site for Shc-There are six tyrosines within the IL-2R␤ chain cytoplasmic domain that upon phosphorylation may serve as binding sites for SH2 domain-containing proteins such as Shc (Fig. 5). Four of these are found within the acid-rich domain. To further clarify which of these phosphotyrosine-containing motifs is involved in mediating Shc binding, COS cells were cotransfected with a hemagglutinin-tagged Shc (Shc-HA) construct, wild type IL-2R␤, and IL-2R␤ constructs containing point mutations of tyrosine to phenylalanine, and a construct encoding the tyrosine kinase Jak-1; co-transfection of these constructs results in tyrosine phosphorylation of each of the IL-2R␤ cytoplasmic tyrosine residues in this assay (data not

TABLE I Characteristics of the IL-2R␤ deletion mutants
This is a summary of the characteristics of the IL-2R␤ deletion mutants stably transfected into Ba/F3 cells as described (31). Tyrosine phosphorylation implies the induction of tyrosine phosphorylation of multiple substrates upon IL-2 stimulation. Induction of Jak-3 activity was measured by changes in tyrosine phosphorylation. Nuclear complex formation was determined by gel mobility shift assay of nuclear extracts using a GRR sequence (31). IL-2-dependent proliferation was measured by [ 3 H]thymidine incorporation (35) in medium supplemented with serum.

IL-2-dependent induction
Tyrosine phosphorylation Jak-3 activation Nuclear complex formation shown). Following transfection and culture, the IL-2R␤ chain was immune precipitated, and Shc association was determined by immunoblotting using an anti-HA antibody. Two conclusions can be drawn from an analysis of the results of a cotransfection experiment of this type presented in Fig. 6. First, Shc is found associated with the IL-2R␤ only in the presence of expressed Jak-1 (Fig. 6, upper panel, and data not shown). This supports our previous observations, which have suggested that IL-2-dependent tyrosine phosphorylation of the ␤ chain is a prerequisite for Shc association. Additionally, this analysis shows that phosphorylated tyrosine 1 at amino acid position 338 within the cytoplasmic domain is required for Shc association (Fig. 6, upper panel). In all transfections, equivalent expression of the IL-2R␤ chain and Shc-HA was confirmed by immunoblot with specific antibody (Fig. 6, middle and lower  panel).

IL-2-dependent Mitogenesis Does Not Require Shc Phosphorylation and Is Supported in the Absence of Ras Activation or
Serum or Insulin Supplements-Previous analysis has suggested that the acid-rich domain of the IL-2R␤ chain is required for IL-2-dependent Ras activation (38). Our results support these observations and further suggest that Shc phosphorylation and Ras activation are not required for IL-2induced mitogenesis in the presence of serum-containing me-dium. This is suggested by the ability of the AD receptor mutants to support IL-2-dependent growth but fail to allow IL-2dependent tyrosine phosphorylation of Shc (Ref. 38 and Fig. 3).
This observation is in sharp contrast to several reports that have clearly shown that the tyrosine phosphorylation of Shc and downstream activation of Ras are closely coupled to ligandinduced mitogenesis (14,39). For example, mutant granulocyte macrophage colony-stimulating factor receptors that do not signal through Ras pathways have been shown to require serum for granulocyte macrophage colony-stimulating factor-induced proliferation (39). The addition of serum in this system results in supplemental Ras activation such that these cells can now respond mitogenically to granulocyte macrophage colonystimulating factor (39). To determine whether serum supplementation is required for IL-2-induced mitogenesis of Ba/F3 cells containing the AD receptor mutant, the proliferative response to IL-2 in wild type (FL) and AD-containing cells under serum-free conditions in the presence or the absence of insulin was analyzed. Although we have previously described minor differences in the IL-2-induced proliferative response of FL and AD receptor-containing cells (31), these results show that FL and AD mutants support increased DNA synthesis (Fig. 7) as well as increases in cell number (data not shown) in response to IL-2 in both serum-free and insulin-free conditions. Although mitogenesis is enhanced in the presence of insulin in these cells, there is no significant difference between FL and AD receptors. The AD mutants fail to activate Ras (38) and fail to activate the Shc adapter pathway, which may result in the activation of other Ras-like pathways, but are capable of supporting IL-2-induced mitogenesis in the presence of serum or in serum-free and insulin-free conditions in a manner similar to that observed with the wild type FL receptors. These results thus suggest that IL-2 signal transduction leading toward mitogenesis does not require Shc phosphorylation or the activation of Shc/Grb2 or Ras pathways (38).

DISCUSSION
Using mutational analysis of the IL-2R system, we have shown that IL-2 induction of Shc tyrosine phosphorylation is dependent on Shc association with the IL-2R␤. We further show that this association is dependent on the presence of phosphorylated tyrosine 1 at amino acid 338 within the acidrich region of the IL-2R␤ cytoplasmic domain. This tyrosine is found within a motif containing the sequence TNQGpYFFF. Structural studies of motifs required for Shc binding to various receptor complexes (40 -42) have revealed a consensus binding site for the Shc phosphotyrosine binding domain (40), also referred to as the SAIN (Shc and IRS-1 NPXY binding) domain (42) of NPXpY. Considering the structural similarities between proline and glutamine, the motif within the IL-2R␤ chain of NQGY appears quite similar to the predicted SAIN domain recognition consensus of NPXY. Using the predicted IL-2 receptor binding domain for Shc, NQXY, a search was initiated for proteins containing this motif (43). Generally, this search identified proteins that have been shown to have regulatory functions in various signal transduction pathways including Ras-Gap (motif from amino acid 178 PTNQWYH-), which is involved in the regulation of Ras and Ras-like pathways, and several receptor molecules such as the fibroblast growth factor receptor (motif from amino acid 761 TSNQEYL-). Experiments aimed at investigating the potential role that Shc association with these and other proteins may play in regulating signal transduction are presently underway.
It has been shown (31,38) that deletion of the acid-rich region of the IL-2R␤ chain abrogates the ability of IL-2 to induce Ras activation as well as increased transcription of fos and jun but did not affect IL-2-dependent mitogenesis in the murine IL-3-dependent cell Ba/F3. This analysis did not preclude the possibility of IL-2-dependent activation of Ras-like but Ras-distinct pathways, which would support IL-2-driven cell growth. By focussing on the analysis of IL-2 activation of upstream Ras pathway regulators (Shc adapter pathway), it becomes much easier to define the potential role that these pathways play in IL-2 signal transduction. Results from this study clearly show that Shc phosphorylation that acts to initiate this pathway is not required for mitogenesis. This analysis does not preclude the possibility that Grb2 may interact directly with this receptor complex and function to activate other guanine nucleotide releasing factor activities in the AD mutant, which may lead to mitogenesis. If this were the case, however, one would expect AD receptors to also activate Ras. This clearly does not occur in this mutant (38). Additionally, in the insulin receptor system, which has been shown to associate independently with both Shc and Grb2, Shc binding and tyrosine phosphorylation is the primary mechanism utilized in vivo to bring Grb2 and guanine nucleotide releasing factor activity to the membrane resulting in Ras activation (44). These data thus suggest that Shc binding to the IL-2R␤ chain, which results in its tyrosine phosphorylation and initiation of the Shc/Grb2 adapter pathway, is the mechanism used by IL-2 to activate Ras and Ras effector pathways.
These data also strongly suggest that IL-2 receptor signal transduction functions to activate specific mitogenic signals without a strict requirement for Ras in IL-2 receptor-containing Ba/F3 cells. This does not preclude the possibility, however, that in the context of a different cellular background IL-2 may have different requirements for Ras and Ras signaling pathways.
It is well described that Ras activation and regulation of downstream pathways is critical in controlling proliferation (14). It seems plausible therefore that mitogenic signal transduction by IL-2 in Ba/F3 cells may activate one or more of these downstream pathways by functionally bypassing Ras activation. A key mitogenic pathway activated by upstream activation of Ras is the Raf/Map kinase cascade (45,46). This pathway has been described as a "mitogenic bottleneck" due to the wide variety of mitogenic signals that funnel into Raf activation (46) and because antisense elimination of Raf blocks cytokine-triggered mitogenesis (47). IL-2 has been shown to activate Raf, and Raf has been found to be associated with the IL-2 receptor complex by ourselves (data not shown) and others (48,49). Furthermore, IL-2-dependent activation of Raf involves a tyrosine kinase-dependent mechanism (48). As has been shown in other systems, this is presumed to involve the tyrosine phosphorylation of Shc, the subsequent activation of Ras, and the downstream activation of Raf and Map kinases (14,46). However, Raf can also be activated by direct tyrosine phosphorylation (50). The possibility exists, therefore, that IL-2 signaling may involve the direct tyrosine phosphorylation of Raf as an activating mechanism. This would theoretically abrogate the requirement for Ras activation in an IL-2-driven mitogenic response. To further elucidate this, the role that Raf activation plays in IL-2 signal transduction in IL-2 receptor mutants is being investigated.
With the data presented here, a potentially novel mechanism by which IL-2, and perhaps other IL-2R␤ chain containing receptors, controls cell growth is being established. In contrast to the results presented from studies within the granulocyte macrophage colony-stimulating factor receptor system (39), which clearly establishes an absolute requirement for some level of Ras activation in order for this receptor system to support cytokine-induced mitogenesis, the model of IL-2 signal transduction that has been generated in Ba/F3 cells establishes that mitogenesis can be fully supported by IL-2 receptors that lack the ability to activate this or similar pathways through FIG. 7. IL-2-induced mitogenesis in FL and AD receptor-containing Ba/F3 cells does not require serum or insulin. Ba/F3 cells containing either the full-length wild type IL-2R␤ (FL) or the acid-rich domain deletion mutant (AD) were treated with 50 ng/ml IL-2 in serumfree medium with or without insulin. Proliferation was measured by [ 3 H]thymidine incorporation. Note that there is no significant difference between FL or AD receptors regarding their ability to transduce IL-2-dependent mitogenic signal in Ba/F3 cells. utilization of the Shc adapter pathway. IL-2 signal transduction thus forms a novel paradigm for mitogenic signaling by hematopoietic cytokines.