Critical role of the membrane-proximal, proline-rich motif of the interleukin-2 receptor gc chain in the Jak3-independent signal transduction Shiho Tsujino1,2, Tadaaki Miyazaki1,3, Atsuo Kawahara3, Michiyuki Maeda4, Tadatsugu Taniguchi1,3 and Hodaka Fujii1,3* 1
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan 2 Department of Hematology and Oncology, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan 3 Institute for Molecular and Cellular Biology, Osaka University, Suita-shi, Osaka 565-0871, Japan 4 Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Abstract Background: The interleukin-2 receptor (IL-2R) consists of three subunits, the IL-2Ra, IL-2Rbc, and IL-2Rgc chains. The essential role of the IL-2Rgc cytoplasmic domain, consisting of 86 amino acids, in signal transmission has been well documented. Particularly, the carboxyl ter-minal region containing 48 amino acids. is essential for the association with and activation of the Jak3 protein tyrosine kinase. On the other hand, little is known about the role of the rest of the IL-2Rgc cytoplasmic region consisting of the membrane-proximal 38 amino acids. Results: We show that a truncated mutant form of IL2Rgc which lacks the membrane-distal 48 amino acids is still capable of inducing the activation of Jak1 and Stat3/Stat5 in the absence of Jak3 activation.
Introduction Interleukin-2 (IL-2) plays a critical role in the immune response by inducing the proliferation and differentiation of lymphocytes. The functional high-affinity IL-2 receptor (IL-2R) consists of three subunits; the IL-2Ra, IL-2Rbc, and IL-2Rgc chains (Smith 1988; Waldmann 1989; Leonard et al. 1994; Minami et al. 1994; Taniguchi 1995; Miyazaki & Taniguchi 1996; Sugamura et al. 1996). The latter two subunits also function as common subunits of other cytokine Communicated by: Kohei Miyazono * Correspondence: E-mail:
[email protected] q Blackwell Science Limited
This membrane-proximal region can also mediate the IL-2-induced tyrosine phosphorylation of the p85 subunit of phosphatidylinositol-3-kinase (PI3K). Furthermore, these signalling events are completely abrogated when mutations are introduced into the proline-rich motif in this region. Conclusions: In this study, we identified a Jak3independent signalling pathway(s) from the membrane-proximal region of IL-2Rgc. Our results indicate that the proline-rich motif in this region plays a critical role in this signalling pathway(s). The present study may provide further insight into the mechanism of cellular responses mediated by IL-2 and other cytokines which utilize the IL-2Rgc for their signal transmission.
receptors, i.e. IL-2Rbc for IL-15R (Grabstein et al. 1994), and IL-2Rgc for IL-4R, IL-7R, IL-9R and IL-15R (Giri et al. 1994; Leonard et al. 1994; Sugamura et al. 1996). The importance of IL-2Rgc in lymphocyte development and function has been underscored by the discovery of mutations in the IL-2Rgc gene in patients with X-linked severe combined immunodeficiency (XSCID), which is characterized by an absence or diminished numbers of T cells (Noguchi et al. 1993). Consistently, mice carrying a null-mutation in the IL-2Rgc alleles also show severe defects in the development of lymphoid cells (Cao et al. 1995; DiSanto et al. 1995). It has been shown that the ligand-induced Genes to Cells (1999) 4, 363–373
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βc
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P266 P269 Jak3
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H
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heterodimerization of the cytoplasmic domains of the IL-2Rbc and IL-2Rgc chains is essential for IL-2induced transmission of the proliferative signals (Kawahara et al. 1994; Nakamura et al. 1994; Nelson et al. 1994). Three subregions that perform distinct signalling functions have been identified within the IL-2Rbc chain. The membrane-proximal, serine-rich region (S-region) is critical for association with the protein tyrosine kinase (PTK) Jak1 (Miyazaki et al. 1994) and Syk (Minami et al. 1995) and for cell proliferation (Hatakeyama et al. 1989). The S-region is also required for the activation of phosphatidylinositol-3-kinase (PI3K) (Merida et al. 1993; Kanazawa et al. 1994). The acidic region (A-region), adjacent to the S-region, is required for the association with and activation of srcfamily PTKs and other signalling molecules such as Shc (Hatakeyama et al. 1991; Kobayashi et al. 1993; Minami et al. 1993; Friedmann et al. 1996; Ravichandran et al. 1996). It has been demonstrated that the S-region mediates IL-2-induced expression of proto-oncogenes such as c-fos, c-jun, c-myc and bcl-2, whereas the A-region mediates the induction of c-fos, c-jun, but not c-myc and bcl-2 (Shibuya et al. 1992; Miyazaki et al. 1995). Finally, the carboxyl-terminal 147 amino acids (H-region) are critical for the activation of Stat5 by IL-2 stimulation (Fujii et al. 1995; Lin et al. 1995). Recently, by using an in vivo approach, it has been shown that the 364
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Summary figure The membraneproximal region of IL-2Rgc mediates intracellular signals including the activation of Jak1, Stat3/Stat5 and PI3K in the absence of Jak3 activation. The prolinerich motif in this region plays the critical role in this Jak3-independent signalling pathway (see text for details).
H-region plays a critical role in the development of natural killer (NK) cells and T cells bearing the gd T cell receptor and that the A-region contributes to effective cytotoxic activity of NK cells and down-regulation of the T cell proliferation function (Fujii et al. 1998). In contrast to the IL-2Rbc chain, the functional analysis of the IL-2Rgc cytoplasmic region has thus far been limited. The cytoplasmic domain of IL-2Rgc contains 86 amino acids (Takeshita et al. 1992). This cytoplasmic region is required for the activation of the Jak1 and Jak3 PTKs (Kawahara et al. 1995), Stat5 (Fujii et al. 1995), and the induction of c-fos, c-myc and bcl-2 (Kawahara et al. 1994, 1995). It has been shown that the C-terminal 48 amino acids of the cytoplasmic domain of IL-2Rgc is required for the association and activation of Jak3 PTK (Asao et al. 1994; Boussiotis et al. 1994; Miyazaki et al. 1994; Russell et al. 1994). More recently, evidence has been provided showing that both membrane-proximal and membrane-distal regions are required for mitogenic signalling and activation of Jak3 (Nelson et al. 1996, 1997) and that a truncation mutant of IL-2Rgc, which cannot associate with and activate Jak3, is still capable of invoking some of the intracellular events caused by IL-2 stimulation, albeit to a limited extent (Nelson et al. 1997). However, the mechanism of Jak3-independent signalling mediated by the membrane-proximal region is still unknown. q Blackwell Science Limited
Signalling motif of the IL-2Rgc chain
mechanism of IL-2-induced signal transduction that is mediated by the membrane-proximal proline-rich motif of the IL-2Rgc chain.
Results Activation of Jak1 and Stat3/Stat5 by a mutant IL-2Rgc lacking the Jak3-binding site
Figure 1 (A) A schematic diagram of the IL-2Rgc chain mutants. Wild-type (WT), M1-mutant, and M2-mutant retain 86, 7, and 38 amino acids of the cytoplasmic domain, respectively. (B) Establishment of IL-2Rgc mutant transformants. ED40515(¹) cells were transfected with vectors encoding wildtype or a mutant version of human IL-2Rgc. Hygromycinresistant clones were stained with an antibody specific for the extracellular domain of human IL-2Rgc (thick line) or with medium alone (thin line) and analysed by flow cytometry. Fluorescence intensity is plotted on the abscissa on a logarithmic scale, and cell number is plotted on the ordinate.
In the present study, we show that a truncated form of IL-2Rgc lacking the carboxyl-terminal 48 amino acids still retains the ability to transduce signals including the activation of Jak1 and Stat3/Stat5 in the absence of Jak3 activation. Furthermore, this membrane-proximal region can also induce tyrosine phosphorylation of p85 subunit of PI3K. Interestingly, these events are completely abolished by introducing mutations into the proline-rich motif, demonstrating the critical role of this proline-rich motif in Jak3-independent signal transmission. Collectively, these results suggest a novel q Blackwell Science Limited
In order to study the function of the cytoplasmic subregions of IL-2Rgc in IL-2-induced signal transduction, we generated deletion mutants of the human IL-2Rgc (Fig. 1A). The M1-mutant lacks the C-terminal 79 amino acids of the cytoplasmic domain of IL-2Rgc, and exerts dominant negative effects when transfected into the IL-2R-reconstituted BAF-B03-derived cell line (Kawahara et al. 1994). The M2-mutant lacks the C-terminal 48 amino acids which are critical for the association of Jak3 with IL-2Rgc (Miyazaki et al. 1994). These mutant and wild-type cDNAs were each inserted into an expression vector pEF (Kawahara et al. 1994), and then transfected into ED40515(¹) cell line (referred to as ED cells hereafter) together with the hygromycin-resistance gene. ED cells are a HTLV-Itransformed human CD4 (þ) CD8 (¹) T cell line, which expresses the IL-2Ra and IL-2Rbc chains but not the IL-2Rgc chain and proliferates independently of IL-2 (Maeda et al. 1985; Arima et al. 1992; Asao et al. 1994). Several hygromycin-resistant clones were assessed for surface expression of wild-type or mutant IL-2Rgc by flow cytometric analysis (Fig. 1B). In view of accumulating evidence indicating critical role for the Jak1 and Jak3 PTKs in IL-2 signalling (Kawahara et al. 1995; Miyazaki et al. 1994), we first examined tyrosine phosphorylation of these PTKs using transformants expressing the wild-type or either of the above IL-2Rgc mutants. It has been demonstrated that enzymatic activities of Jak1 and Jak3 are well correlated with their phosphorylation levels (Boussiotis et al. 1994; Johnston et al. 1994; Miyazaki et al. 1994; Russell et al. 1994; Witthuhn et al. 1994). In WT-24 cells expressing the cDNA-encoded wild-type IL-2Rgc, both Jak1 and Jak3 are activated upon IL-2 stimulation (Fig. 2A,B), whereas none of these PTKs was activated in the parental ED cells (data not shown). On the other hand, these PTKs are not activated in M1–4 cells expressing the M1-mutant (Fig. 2A,B), confirming the essential role of the cytoplasmic region of IL-2Rgc (Kawahara et al. 1994). As expected from previous experiments (Miyazaki et al. 1994; Russel et al. 1994; Nelson et al. 1996, 1997), IL-2-induced activation of Jak3 was not detectable in M2-7 cells, which express an IL-2Rgc Genes to Cells (1999) 4, 363–373
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Figure 2 Activation of Jaks and Stats in transformant clones expressing IL-2Rgc mutants. (A) The membrane-distal region is necessary for Jak3 activation. (B) The membrane-proximal region can mediate Jak1 activation by IL-2. Cells were stimulated or mockstimulated with 2 nM IL-2 for 10 min. Cells were lysed, and Jak3 (A) or Jak1 (B) was immunoprecipitated with specific anti-serum. Immunoprecipitates were separated by SDS-PAGE and subjected to immunoblot analysis with an anti-phosphotyrosine antibody (4G10) (Upper). Jak kinase protein levels were determined by immunoblot with the respective Jak anti-serum. We confirmed that activation of Jak3 was not detected even after longer exposure of the blot. (C) The membrane-proximal region can induce Stat activation in the absence of Jak3 activation. Cells were stimulated or mock-stimulated with 2 nM IL-2 for 10 min. Whole-cell extracts were prepared and incubated with 32P-labelled IRF-1 GAS probe. Complexes were resolved by separation on 4% polyacrylamide gels and detected by autoradiography. (D) WT-24 cells were stimulated with 2 nM IL-2. Before incubation with 32P-labelled IRF-1 GAS probes, extracts were incubated for 1 h at 4 8C with nonimmune serum (NIS), anti-Stat1 antibody, anti-Stat3 antibody or anti-Stat5 antibody.
mutant lacking the C-terminal 48 amino acids (i.e. the Jak3-binding site) (Miyazaki et al. 1994). Interestingly, it was found that IL-2 induced activation of Jak1 PTK in M2-7 cells (Fig. 2A, B). Notably, Jak1 PTK activation in this cell line was much lower (about fivefold) than that in WT-24 cells, suggesting that Jak3 also contributes to full activation of Jak1. Similar observations were made in other independently isolated transformant cells expressing the same cDNA (data not shown). These observations indicate that Jak1 PTK can be activated following IL-2 stimulation in the absence of Jak3 PTK activation, albeit at a lower level, and suggest that the membrane-proximal region may play a novel role in this process. It has been shown that IL-2R stimulation results in the activation of Stat3 and Stat5 by Jak1- and 366
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Jak3-mediated tyrosine phosphorylation. We then examined the activation of these Stat proteins in these transformant cell lines by electrophoretic mobility shift assay (EMSA). As shown in Fig. 2C, DNA binding complexes were detected following IL-2 stimulation in cells expressing the wild-type IL-2Rgc, WT-17 and WT-24 cells. These complex were partially supershifted by an anti-Stat5 antibody, and its activity was diminished by an anti-Stat3 antibody, suggesting that both of Stat5 and Stat3 had been activated (Fig. 2D). The activation of Stat proteins was not detected in cells expressing the M1-mutant, M1-4 and M1-18 (Fig. 2C). Interestingly, the activation of these Stats was also observed in cells expressing the M2-mutant, M2-7 and M2-20, although the level of activation was weaker than that of WT-17 and WT-24 cells (Fig. 2C). These results q Blackwell Science Limited
Signalling motif of the IL-2Rgc chain
cells, although the phosphorylation level was significantly lower than that observed in WT-24 cells (Fig. 3A). These results indicate that the membrane-proximal region of IL-2Rgc can mediate IL-2-induced tyrosine phosphorylation of the p85 subunit of PI3K without activating Jak3. It has been reported that IL-2 stimulation induces MAPK activation (Karnitz et al. 1995; Monfar et al. 1995). We found that the activation of MAPK by IL-2 occurred in WT-24 cells, but did not occur in M2-7 cells (Fig. 3B). These results suggest that, unlike PI3K activation, the MAPK activation by IL-2 is totally dependent on the carboxyl-terminal 48 amino acids of IL-2Rgc, which is essential for Jak3 activation.
Critical role of the proline-rich motif in the membrane-proximal region of IL-2Rgc
Figure 3 (A) The membrane-proximal region of the IL-2Rgc can induce the activation of PI3K. Cells were mock-stimulated or stimulated with 2 nM IL-2. Cells were lysed, and the 85 kDa subunit of PI3K was immunoprecipitated with anti-serum to p85. The immunoprecipitates were then resolved by SDS-PAGE and analysed by immunoblotting with anti-phosphotyrosine (4G10) (Upper). Protein levels of p85 were determined by antip85 anti-serum. (B) The membrane-distal region is required for the activation of MAPK. Cells were mock-stimulated or stimulated with 2 nM IL-2 and were lysed. Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting with anti-active MAPK antibody (upper). Protein levels were monitored by anti-MAPK anti-serum (lower).
indicate that activation of Stats by IL-2 can occur in the absence of Jak3 PTK activation.
Phosphorylation of the p85 subunit of PI3K by the membrane-proximal region of IL-2Rgc The above observations prompted us to examine whether activation of other signalling molecules can be mediated by the IL-2Rgc mutant lacking the Jak3 association (the M2-mutant). It has been shown that PI3K is activated following IL-2R stimulation (Augustine et al. 1991; Merida et al. 1991; Remillard et al. 1991). Consistently with these reports, the IL-2 induction of tyrosine phosphorylation of the p85 subunit of PI3K was observed in WT-24 cells (Fig. 3A). Interestingly, p85 phosphorylation by IL-2 was also observed in M2–7 q Blackwell Science Limited
It has been pointed that a certain homology exists among the membrane-proximal region of IL-2Rgc and those of other cytokine receptors, i.e. the a-chains of the GM-CSFR, IL-3R, and IL-5R (Takaki et al. 1994; Nelson et al. 1996). This ‘proline-rich motif ’ is also homologous to the corresponding regions of the prolactin receptor and growth hormone receptor (Fig. 4A) (Nelson et al. 1996). It was shown that the proline residues P352 and P356 in IL-5Ra are critical for IL-5-mediated cell growth and Jak1/Jak2 PTK activation (Cornelis et al. 1995; Kouro et al. 1996). To examine the importance of this proline-rich motif in IL-2Rgc in signal transduction mediated by its membrane-proximal region, we substituted alanine residues for P266 and P269 in the M2-mutant of IL-2Rgc to construct the PA-mutant variant (Fig. 4B). The expression vector encoding the mutant cDNA was transfected into ED cells, and stable transformed clones were established (Fig. 4C). In cells expressing the PA-mutant (PA-2 cells), activation of Jak1 and Stats by IL-2 was completely abolished (Fig. 5A, B). In addition, tyrosine phosphorylation of p85 subunit of PI3K was not detected in PA-2 cells (Fig. 5C). Similar observations were made in independently isolated transformant clones, each expressing the same cDNA (data not shown). These data suggest that the proline-rich motif of IL-2Rgc plays a critical role in the IL-2 signalling mediated by the membrane-proximal region of IL-2Rgc.
Discussion The functional high-affinity IL-2 receptor (IL-2R) consists of three subunits; the IL-2Ra, IL-2Rbc, and Genes to Cells (1999) 4, 363–373
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Figure 4 (A) Sequence alignment of the cytoplasmic domains of the human IL2Rgc, IL-3Ra, IL-5Ra, GM-CSFRa, prolactin receptor (PRLR), and growth hormone receptor (GHR). Conserved and related amino acid residues are shown in the lower. (B) A schematic diagram of the IL-2Rgc point mutants. The PA-mutant retains 38 amino acids of the cytoplasmic domain. Positions of proline residues replaced with alanines are indicated. (C) Establishment of the IL-2Rgc PA-mutant transformants. Procedures are the same as described in Fig. 1B.
IL-2Rgc chains (Leonard et al. 1994; Minami et al. 1994; Sugamura et al. 1996). It has been shown that the ligand-induced heterodimerization of the cytoplasmic domains of the IL-2Rbc and IL-2Rgc chains is essential for IL-2-induced transmission of the proliferative signals (Kawahara et al. 1994; Nakamura et al. 1994; Nelson et al. 1994). Although IL-2Rbc and IL-2Rgc contain no catalytic motifs such as the PTK motif within their cytoplasmic regions, they can recruit multiple PTKs as well as other signalling molecules so as to facilitate the diverse actions of cytokines on various target cells (Ihle 368
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1995; Taniguchi 1995). Thus far, it has been well established that specific signalling molecules are linked to specific subregions of IL-2Rbc (see Taniguchi 1995, for review), and roles of each subregion in lymphocyte proliferation and differentiation have been extensively examined in vitro (see Taniguchi 1995; Sugamura et al. 1996; for reviews) and in vivo (Fujii et al. 1998). In contrast to the IL-2Rbc chain, the functional analysis of the IL-2Rgc cytoplasmic region has thus far been limited. In this study, we expressed various mutants of q Blackwell Science Limited
Signalling motif of the IL-2Rgc chain
Figure 5 The proline residues of the membrane-proximal domain of IL-2Rgc are critical for signal transduction mediated by the membrane-proximal region of IL-2Rgc. (A) Tyrosine phosphorylation of Jak1 in response to IL-2. Procedure was the same as described in Fig. 2A. (B) Stat activation by IL-2. EMSA was performed as described in Fig. 2C. (C) Tyrosine phosphorylation of p85 by IL-2. Procedure was the same as described in Fig. 3A.
IL-2Rgc in the IL-2Rgc-negative human T cell line ED in order to functionally dissect its cytoplasmic domain. Previously, it was shown by a transient expression system in nonlymphoid cells that deletion of the C-terminal 48 amino acids of IL-2Rgc abrogates its association with Jak3 (Miyazaki et al. 1994). q Blackwell Science Limited
Consistent with this, the activation of Jak3 was completely abolished in M2-7 cells, an ED-derived clone expressing the corresponding mutant receptor (Fig. 2A). On the other hand, we found that tyrosine phosphorylation of Jak1 by IL-2 is still induced in these cells (Fig. 2B). These results suggest that the basal activation of Jak1 by IL-2 is induced independently of the activation of Jak3. It is an interesting issue how Jak1 can be activated by IL-2 in the absence of Jak3 activation, and we demonstrated that the proline-rich motif in the membrane-proximal region of IL-2Rgc plays a critical role in this process (see below). We further showed that activation of Stat3/Stat5 and tyrosine phosphorylation of p85 subunit of PI3K were also observed in M2–7 cells (Figs 2C and 3A). It is most likely that the activated Jak1 is responsible for these signalling events in the absence of Jak3 activation. Interestingly, tyrosine phosphorylation of Jak1 is significantly weaker in M2-7 cells than in WT-24 cells. In this regard, it has been reported that expression of a dominant negative mutant of Jak3 significantly inhibited the IL-2-induced activation of Jak1 (Kawahara et al. 1995). These observations are both consistent with the idea that Jak3 contributes to full activation of Jak1. Activation of Stats and tyrosine phosphorylation of p85 subunit of PI3K are also diminished in M2-7 cells, as compared with those in WT-24 cells. These results indicate that Jak3 augments IL-2-induced signal transduction by enhancing the activity of Jak1 as well as directly phosphorylates signalling molecules. In this regard, it should be noted that DNA binding activity of Stats increased significantly by ectopic expression of Jak3 in IL-2R-reconstituted fibroblast cell lines (Fujii et al. 1995). In addition, direct involvement of Jak1 and Jak3 in PI3K activation has also been suggested (Johnston et al. 1995). On the other hand, activation of MAPK is totally dependent on membrane-distal region of IL-2Rgc (Fig. 3B), suggesting a critical role of Jak3 in MAPK activation. One candidate molecule which links Jak3 to MAPK is Pyk2 PTK, which leads to activation of MAPK through G-protein-coupled receptors and by stress signals (Dikic et al. 1996). In this context, it has been recently shown that Pyk2 is activated by IL-2 stimulation, and Pyk2 activation by IL-2 was totally dependent on Jak3 (Miyazaki et al. 1998). In fact, Pyk2 activation by IL-2 was not detected in M2-7 cells (Miyazaki et al. 1998). Recently, the function of the membrane-proximal region of IL-2Rgc was also analysed by using mouse CTLL-2 cells expressing chimeric receptors, in which the GM-CSF receptor b or a extracellular regions were fused to the IL-2Rbc or IL-2Rgc cytoplasmic regions, Genes to Cells (1999) 4, 363–373
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respectively (Nelson et al. 1997). It was found that the membrane-proximal 39 amino acids region of IL-2Rgc mediates phosphorylation of the tyrosine phosphatase SHP-2 and IL-2Rbc in the absence of Jak3 activation. However, in their experiments, activation of Jak1 was not detected, an observation which is not consistent with our present findings (Fig. 2). Since our data showed that the activation level of Jak1 was significantly reduced in the absence of Jak3 activation, it is possible that Jak1 activation by the mutant receptor was under threshold of detection in the CTLL-2 system. It is also possible that the fusion of GM-CSFR and IL-2Rgc affected the conformation of the proximal-region to lose its ability to activate Jak1. Alternatively, there may exist cell-specific mechanism which is required for IL-2-induced Jak1 activation. This point will require further clarification. In this study, we have shown that the proline-rich motif of the membrane-proximal domain of IL-2Rgc is critical for Jak3-independent signal transmission. In fact, the IL-2-induced phosphorylation of Jak1 and PI3K p85 subunit and activation of Stat proteins observed in M2-7 cells were completely abolished in PA-2 cells (Fig. 5). Interestingly, a similarity has been noted between this membrane-proximal region of the IL-2Rgc and other cytokine receptor subunits (Nelson et al. 1996), and the corresponding proline residues in other cytokine receptors have been shown to be critical for signal transduction (Takaki et al. 1994; Cornelis et al. 1995; Kouro et al. 1996). An interesting issue for future study is the question of how this proline-rich membrane-proximal region transmits intracellular signals. One attractive possibility is that some signalling molecule(s) is associated with this portion of IL-2Rgc. In fact, the critical proline-rich sequence of the IL-2Rgc contains a motif, P-X-X-P, that is conserved among various Src homology 3 domain-binding proteins (Ren et al. 1993; Yu et al. 1994). In this regard, it is interesting to recall that expression of a dominant interfering mutant of Jak3 in BAF-B03derived cells results in quasi-complete inhibition of the endogenous Jak3 activation by IL-2, but that induction of bcl-2 gene by IL-2 was observed (Kawahara et al. 1995). Thus, an intriguing possibility is that the Jak3independent bcl-2 induction pathway may be linked to this region, and this possibility is currently examined in normal lymphocytes by expressing the M2-mutant cDNA in mice carrying an IL-2Rgc null mutations. On the other hand, an alternative possibility, which cannot be excluded at this stage, is that interaction of this membrane-proximal domain with Jak1 or IL-2Rbc may itself be critical for the signal transduction. 370
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Since ED cells proliferate independently of IL-2 (Arima et al. 1992), it is difficult to address the physiological significance of signals mediated by the membrane-proximal region of IL-2Rgc in the experimental system used in the present study. In order to assess this issue, work is in progress to express IL-2Rgc mutant cDNAs on an IL-2Rgc null background. In our preliminary experiments, lymphocyte development abrogated in IL-2Rgc null mice was partially restored by the expression of a murine version of the M2-mutant used in this study, indicating potential importance of the signals mediated by the membrane-proximal region in vivo (S.T., T.T. and H.F., unpublished observation). Our results in this study may provide the molecular basis for such in vivo analyses.
Experimental procedures Cell culture ED40515(¹) is a HTLV-I-transformed human CD4(þ) T cell line (Maeda et al. 1985; Asao et al. 1994) and was maintained in RPMI 1640 medium supplemented with 10% (v/v) FCS. ED40515(¹)derived cell lines were maintained in the presence of 0.5 mg/mL of hygromycin (Wako Pure Chemicals, Osaka, Japan).
Plasmid construction and DNA transfection Construction of expression vectors of wild-type (pEFIL2Rg) and M1-mutant lacking the C-terminal 79 amino acids of the human IL-2Rgc chain (pEFIL2RgM1) was described previously (Kawahara et al. 1994). For the construction of the expression vector for the M2-mutant which lacks the C-terminal 48 amino acids of the human IL-2Rgc chain (pEFIL2RgM2), pIL-2Rg2 (Takeshita et al. 1992) was amplified by PCR using synthetic oligonucleotides 50 -TGGCTCCATGGGATTGATTATCAG-30 and 50 -TGGGGTACCTCTAGAGACTCTCAGCCAGTCCCT TAGACAC-30 as primers. The PCR product was digested with KpnI and NcoI, and ligated with NcoI/KpnI-digested pIL-2Rg2. The mutant IL-2Rgc cDNA was excised by XbaI and ligated with XbaI-cleaved pEF expression vector (Kawahara et al. 1994). For the construction of expression vector for PA-mutant (pEFIL2RgPA), a portion of the membrane-proximal cytoplasmic region of pIL-2Rg2 was amplified using oligonucleotides 5 0 -AAGCCGTGGTTATCTCTGTT-3 0 (P1) and 50 -TGGCAATTCGGGCCATCGTCCGTTC-30 (P2) primers, and another portion was amplified using oligonucleotides 50 -ATGGCCCGAATTGCCACCCTGAAGA-30 (P3) and 50 -CTGTCTAGAGACTCTCAGCC-30 (P4) primers. The second PCR was performed with P1 and P4 primers, using the first PCR product as a template. The second PCR product was digested with NcoI and XbaI, and ligated with HindIII/NcoI fragment of pIL-2Rg2 and pBluescriptSK(þ) cleaved with HindIII and XbaI. The cDNA for the PA-mutant was excised with EcoRI and XbaI, and then ligated into the pEF vector
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Signalling motif of the IL-2Rgc chain cleaved with EcoRI and XbaI. All constructs were confirmed by restriction enzyme digestion and DNA sequencing. Plasmid DNAs were transfected into the cells by an electroporation procedure as described previously (Hatakeyama et al. 1989). Selection was initiated 24 h after transfection, using 0.5 mg/mL of hygromycin. Drug-resistant single colonies were obtained as described previously (Hatakeyama et al. 1989).
Flow cytometric analysis Cells (,1 × 106) were treated with phycoerythrin (PE)-conjugated mouse monoclonal antibody TUGh4 (Pharmingen, San Diego, CA) (Asao et al. 1994) recognizing the extracellular domains of the human IL-2Rgc chain for 30 min at 4 8C. After washing, the stained cells were analysed by FACS Caliber Flow Cytometer (Beckton-Dickinson, San Jose, CA).
Immunoprecipitation and immunoblot analysis Immunoprecipitation and immunoblot analyses were done as described (Miyazaki et al. 1994). Rabbit anti-human Jak1 antiserum, rabbit anti-human Jak3 anti-serum, mouse monoclonal anti-phosphotyrosine antibody (4G10) and rabbit anti-PI3K p85 subunit anti-serum were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-active MAPK anti-serum was purchased from Promega (Madison, WI). Rabbit anti-MAPK anti-serum was provided by Drs Ueki, Tobe and Kadowaki.
Electrophoretic mobility shift assay (EMSA) Cells were treated with 2 nM IL-2 for 10 min. Preparation of whole cell extract, binding reaction, and gel electrophoresis were done as described (Fujii et al. 1995). The oligonucleotide sequence was derived from the mouse IFN regulatory factor-1 (IRF-1) GAS (IFNg activated site) (50 -GCCTGATTTCCCC GAAATGATGA-30 ) (Sims et al. 1993). For supershifts, control antibody, anti-human Stat1 antibody (Transduction Laboratories, Lexington, KY), anti-mouse Stat3 (Santa Cruz Biotechnology, Santa Cruz, CA) or chicken anti-sheep Stat5 anti-serum (provided by Dr B. Groner) (Fujii et al. 1995) was preincubated with cell extracts for 1 h at 4 8C before probe addition.
Acknowledgements We thank Drs G. Harris and M. S. Lamphier for critical reading of the manuscript. We are grateful to Dr K. Sugamura for providing the human IL-2Rgc plasmid (pIL-2Rg2), Drs Ueki, Tobe, and Kadowaki for rabbit anti-MAPK antiserum, Dr B. Groner for anti-Stat5 anti-serum. We are also grateful to Drs B. H. Nelson and P. D. Greenberg for sharing with us their unpublished results. This work was supported in part by the Research for the Future (RFTF) Program (96L00307) from the Japan Society for the Promotion of Science (JSPS) (to T.T.), and a Grant-in-Aid for Scientific Research on Priority Areas, no. 09273105 (to H.F. and T.M.) from the Ministry of Education, Science, Sports and Culture of Japan.
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Received: 1 April 1999 Accepted: 15 May 1999
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