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The Journal of Immunology, 1999, 162: 1496-1501.
Copyright © 1999 by The American Association of Immunologists

The Mapping of the Lyn Kinase Binding Site of the Common ß Subunit of IL-3/Granulocyte-Macrophage Colony- Stimulating Factor/IL-5 Receptor1

Tetsuya Adachi, Konrad Pazdrak, Susan Stafford and Rafeul Alam2

Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been shown that a membrane-proximal region within common ß (ßc) receptor of IL-3/granulocyte-macrophage CSF/IL-5 (amino acids 450–517) is important for Lyn binding. We have shown previously that Lyn kinase is physically associated with the IL-5R ßc subunit in unstimulated cells. The result suggests that this association involves binding modules that are not activation or phosphorylation dependent. The objective of this study was to map the exact Lyn binding site on ßc. Using overlapping and/or sequential peptides derived from ßc 450–517, we narrowed down the Lyn binding site to nine amino acid residues, ßc 457–465. The P->A mutation in this region abrogated the binding to Lyn, indicating a critical role of proline residues. We created a cell-permeable Lyn-binding peptide by N-stearation. This cell-permeable peptide blocked the association of Lyn, but not Jak2 with ßc in situ. We also investigated the ßc binding site of Lyn kinase. Our results suggest that the N-terminal unique domain of Lyn kinase is important for binding to ßc receptor. To our knowledge, this is the first molecular identification of the Lyn binding site of ßc receptor. This finding may help develop specific inhibitors of Lyn-coupled signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-3, GM-CSF,3 and IL-5 are three important growth and differentiation factors for myeloid progenitors. IL-3 is a pluripotent stem cell growth factor. GM-CSF promotes the growth and differentiation of granulocytes and macrophages. IL-5 is a specific differentiation factor for eosinophils and basophils. Furthermore, IL-5 is important for survival and activation of eosinophils (1). All three cytokines have specific {alpha} receptors, but they share a common ß (ßc) receptor (2). The ßc subunit is the principal signaling receptor and, consequently, all three hematopoietins have significant functional overlap, especially with regard to their activity on eosinophils. The latter cells play a crucial role in the pathogenesis of asthma and allergic diseases (3).

We have shown that the ßc receptor activates Lyn and Jak2 tyrosine kinases and propagates signals through the Ras-Raf 1-mitogen-activated protein kinase and Jak2-STAT pathways in eosinophils (4, 5). Lyn and Jak2 are physically associated with the ßc subunit of the IL-5R (4, 5, 6). A recent study has demonstrated that at least two distinct regions within the cytoplasmic domain of the ßc are responsible for signal transduction. A membrane-proximal region upstream of amino acid 517 is essential for induction of c-myc and pim-1 (7), whereas a distal region between amino acid 545 and 589 is required for activation of c-fos promoter (8). Other investigators have shown that a truncated receptor, ßc 1–517, binds Lyn, Fes, and Jak2 kinases (9). The first 1–449 residues of the receptor constitute the extracellular and transmembrane domain. Thus, it appears that the membrane-proximal 450–517 residues are important for the activation of some critical tyrosine kinases, including Lyn kinase.

The significance of Lyn kinase in eliciting specific cellular functions has been studied. In the Lyn knockout mice, the activation of mast cells is impaired (10, 11). The mice fail to develop passive anaphylactic reactions. The mice demonstrate pancytopenia, and the cause is unclear. In mature human eosinophils, Lyn is essential for the activation of the antiapoptotic pathways (6, 12). However, Lyn does not appear to be not important for eosinophil degranulation or up-regulation of adhesion molecules (12).

The objective of this study was to map the Lyn kinase binding site of ßc receptor. To this goal, we generated sequential and overlapping peptides from the ßc and determined their binding to Lyn kinase in vitro. We demonstrated that a region of the ßc between amino acid 457 and 465 is the critical binding site of Lyn kinase. This discovery of the Lyn binding site may help develop specific inhibitors of the Lyn-coupled signaling pathway and the generation of novel therapeutic modalities for asthma, allergic, and other eosinophilic disorders.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The synthesis of peptides and their biotinylation were performed by Quality Controlled Biochemicals (Hopkinton, MA). The Lyn-truncated GST fusion proteins, Lyn[1–243], Lyn[1–61], Lyn[1–119], and Lyn[131–243], were obtained from PharMingen (San Diego, CA). A TF-1 cell line was purchased from American Type Culture Collection (Manassas, VA). Streptavidin, GST, and peroxidase-conjugated rabbit anti-goat IgG Ab were obtained from Sigma (St. Louis, MO). The rabbit polyclonal anti-Lyn, anti-SHP-2, anti-Jak2, anti-IL-3/IL-5/GM-CSFRß Abs, horseradish peroxidase-conjugated goat anti-rabbit IgG Ab, and protein A/G plus agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-GST Ab was purchased from Pharmacia (Piscataway, NJ). Enhanced chemoluminescence detection system and Hybond ECL nitrocellulose membrane were obtained from Amersham (Arlington Heights, IL).

Peptides

The five biotinylated peptides from the ßc membrane-proximal region (ßc 450–465, YGYRLRRKWEEKIPNP-NH2; ßc 457–465, KWEEKIPNP-NH2; ßc 457–471, KWEEKIPNPSKSHLF-NH2; ßc 462–481, IPNPSKSHLFQNGSAELWPP-NH2; ßc 482–498, GSMSAFTSGSPPHQGPW-NH2) were synthesized. For some experiments, these peptides were modified by N-stearation instead of biotinylation. A mutated ßc 463–482 peptide with P->A substitution was also used for the experiment. The biotinylated and tyrosine-phosphorylated ßc 605–624 (pY612) peptide (PPPGSLEpYLCLPAGGQVQLV-NH2) was used as a positive control. The biotinylated peptides corresponding to the amino acid residue 29–48 of human Fc{epsilon}RIß receptor (EISPQEVSSGRLLKSASSPP-NH2) and 658–677 of human gp130 (PNVPDPSKSHIAQWSPHTPP-NH2), which are located in their membrane-proximal regions, were used for negative controls. The peptides were purified to >95% by HPLC. The purity of the peptides and their modification were judged by mass spectrometry.

Immunoprecipitation

TF-1 cells (106 cells) were incubated with the N-stearated peptides for 2 h at 37°C and lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 µg/ml aprotinin, leupeptin, and pepstatin). After 20 min on ice, detergent-insoluble materials were removed by centrifugation at 4°C at 12,000 x g. The protein concentration was determined using bicinchoninic acid assay (Pierce, Rockford, IL). The cell lysates were precleared by incubation with 20 µl of the protein A/G plus agarose for 30 min. After removal of the beads, the lysates were incubated with the appropriate Ab (1–2 µg for each sample) for 1 h, followed by the incubation with 20 µl of protein A/G plus agarose for 2 h at 4°C. The beads were washed three times with the cold lysis buffer. The immunoprecipitates were boiled in twofold concentrated Laemmli reducing buffer for 2 min.

Peptide-binding assay

TF-1 cells or human blood leukocytes (5 x 106 cells) were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 10% glycerol, 1% Triton X-100, 1 µg/ml aprotinin, leupeptin, and pepstatin. Aliquots of the cell lysate were precleared with avidin-conjugated agarose beads and then incubated with the biotinylated peptides (0.5–50 µM, as indicated) for 4 h. The biotinylated peptide-Lyn complex was precipitated with 20 µl of avidin-conjugated agarose beads for 2 h at 4°C. After the last incubation, the beads were washed five times with lysis buffer and suspended in twofold concentrated Laemmli reducing buffer, followed by boiling for 2 min. In some experiments, the Lyn-truncated GST fusion proteins were used as sources of Lyn instead of the cell lysates.

Gel electrophoresis and immunoblotting

SDS-polyacrylamide gels were prepared according to the Laemmli protocol and used for immunoblotting. The concentration of polyacrylamide was 8%. Gels were blotted onto Hybond membranes for Western blotting using the enhanced chemoluminescence system. Blots were incubated in a blocking buffer containing 10% BSA in TBST buffer (20 mM Tris-base, 137 mM NaCl, pH 7.6, 0.05% Tween-20) for 1 h, followed by incubation in the primary Ab (0.1 µg/ml) for 2 h. After washing three times in TBST buffer, blots were incubated for 30 min with a horseradish peroxidase-conjugated secondary Ab (0.05 µg/ml) directed against the primary Ab. The blots were developed with the enhanced chemoluminescence substrate, according to manufacturer’s instruction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mapping the Lyn binding site of ßc receptor

A previous study showed that a membrane-proximal region of ßc (amino acids 450–517) was critical for the binding of Lyn, Fes, and Jak2 (9). For this reason, we synthesized overlapping and/or sequential peptides (ßc 450–465, ßc 457–471, ßc 462–481, ßc 482–498) derived from this region (Fig. 1Go). A phosphorylated peptide derived from ßc, the ßc 605–624 (pY612) peptide, was used as a positive control because it bound to the Src homology 2 (SH2) domain of Lyn kinase (13). We also obtained two control peptides derived from Fc{epsilon}RIß (amino acids 29–48) and gp130 (amino acids 658–677). Fc{epsilon}RIß and gp130 were chosen as the source of our control peptides because they are known to bind Lyn kinase (14, 15, 16). These particular peptides were selected from Fc{epsilon}RIß and gp130 because they were rich in proline residues and the proline residues formed a specific pattern, P(X)14PP. Therefore, we asked whether this pattern constituted a binding motif for Lyn kinase. ßc 462–481, IPNPSKSHLFQNGSAELWPP; Fc{epsilon}RIß 29–48, EISPQEVSSGRLLKSASSPP; gp130 658–677, PNVPDPSKSHIAQWSPHTPP.



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  		FIGURE 1. A previous study showed that a membrane-proximal region of ßc (amino acids 450–517) was critical for binding of Lyn, Fes, and Jak2. For this reason, five overlapping and/or sequential peptides (ßc 450–465, ßc 457–465, ßc 457–471, ßc 462–481, ßc 482–498) were synthesized from this region.

 
All of these peptides were biotinylated and used in Lyn-binding experiments. Three overlapping peptides (ßc 450–465, ßc 457–471, ßc 462–481), but not ßc 482–498, Fc{epsilon}RIß 29–48, and gp130 658–677 peptides, bound Lyn kinase (Fig. 2GoA). The left lane containing the unprocessed cell lysate shows the position of the p53/p56 Lyn kinase. The lack of Lyn binding to Fc{epsilon}RIß 29–48 and gp130 658–677 peptides suggests that the P(X)14PP pattern does not constitute a binding motif. A previous report indicated that the PXP motif of the so-called box 1 region of gp130 was a Jak kinase binding site (17). Since our Lyn-binding peptides have a PXP motif and the ßc receptor associates with Jak2 kinase, we investigated whether our peptides bound Jak2. We were unable to demonstrate the binding of Jak2 to ßc 462–481 or ßc 450–465 peptides (Fig. 2GoB). The right lane containing the cell lysate immunoprecipitated with anti-Jak2 Ab shows the position of the Jak2 kinase.



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  		FIGURE 2. The binding of the sequential and/or overlapping peptides from the ßc receptor to Lyn (A) and Jak2 (B) kinases. Precleared TF-1 cell lysates were incubated with or without the biotinylated peptides (50 µM) for 4 h, followed by incubation with avidin-agarose for 2 h. The avidin-agarose-bound proteins were Western blotted with anti-Lyn Ab. A, The left lane containing the unprocessed cell lysate shows the position of the p53/p56 Lyn kinase. The ßc 605–624 (pY612) peptide was used as a positive control. Three overlapping peptides (ßc 450–465, ßc 457–471, ßc 462–481), but not ßc 482–498, bound to Lyn kinase. The Fc{epsilon}RIß 29–48 and gp130 658–677 were control peptides (n = 3). B, The right lane containing the cell lysate immunoprecipitated with anti-Jak2 Ab shows the position of the Jak2 kinase. Neither the ßc 450–465 nor the ßc 462–481 peptide bound to Jak2 kinase (n = 3).

 
The ßc 462–481 peptide bound Lyn in a dose-dependent manner (Fig. 3Go). Proline residues have been implicated in hydrophobic interactions (e.g., SH3 binding) among various proteins (18). Our peptides had two to four proline residues in their sequences. To determine the role of the proline residues, we performed experiments with a P->A substituted peptide. The mutated ßc 462–481 peptide with P->A substitution did not bind to Lyn (Fig. 4Go). To further narrow down the binding site for Lyn kinase, we synthesized a nonamer peptide from the ßc (amino acids 457–465) that encompasses the overlapping PXP motif. The ßc 457–465 peptide also bound Lyn kinase (Fig. 5Go). Our results suggest that Lyn binds to the region of ßc 457–465, and that proline residues in this region have a critical role for Lyn binding.



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  		FIGURE 3. The dose-dependent binding of ßc 462–481 peptide to Lyn kinase. Human blood leukocytes were lysed and precleared with avidin-conjugated agarose. The lysates were incubated with increasing concentrations of the biotinylated ßc 462–481 peptide for 4 h, followed by incubation with avidin-agarose for an additional 2 h. The agarose-bound proteins were eluted and Western blotted with anti-Lyn Ab. The left lane containing the unprocessed cell lysate shows the position of the p53/p56 Lyn kinase. The peptide bound the p53 Lyn in a dose-dependent manner (n = 3).

 


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  		FIGURE 4. The binding of Lyn kinase to the mutated ßc 462–481 peptide. TF-1 cell lysates were precleared with avidin-agarose and then incubated with the ßc 462–481 peptide (50 µM) or the mutated ßc 462–481 peptide with P->A substitution (50 µM). After incubation for 4 h, the avidin-agarose conjugates were added and the bound proteins were separated. The proteins were electrophoresed and Western blotted with anti-Lyn Ab. The left lane containing the unprocessed cell lysate shows the position of the p53/p56 Lyn kinase. The blot shows that the mutated ßc 462–481 peptide did not bind to Lyn kinase (n = 3).

 


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  		FIGURE 5. The binding of nonamer ßc 457–465 peptide to Lyn kinase. Pecleared TF-1 cell lysates were incubated with or without the biotinylated peptides (50 µM) for 4 h, followed by incubation with avidin-agarose for 2 h. The avidin-agarose-bound proteins were Western blotted with anti-Lyn Ab. The ßc 457–465 peptide bound to Lyn kinase (n = 3).

 
Inhibition of physical association of Lyn with ßc in situ by a N-stearated Lyn-binding peptide

To date, we have shown that ßc-derived peptides encompassing the PXP motif (residues 463–465) bind Lyn in vitro. If these peptides bind Lyn in situ, they should block physical association of the latter with ßc receptor. To this goal, we first created a cell-permeable peptide by N-stearation. N-acylation of small peptides has recently been shown to cause their internalization through the lipid membranes, as demonstrated by spin label electron spin resonance and two-dimensional nuclear magnetic resonance techniques (19). The N-myristoylation of a protein kinase C substrate peptide analogue causes its internalization and specific inhibition of the kinase (20). TF-1 cells were incubated with the N-stearated peptide for 2 h and then lysed. The cell lysates were immunoprecipitated with anti-Lyn or anti-Jak2 Ab, followed by electrophoresis and Western blotting with anti-ßc Ab. The coprecipitation of ßc with Lyn, but not with Jak2, was inhibited by the N-stearated Lyn-binding peptide in a dose-dependent manner (Fig. 6Go), indicating a specific binding of the peptide to Lyn kinase in situ.



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  		FIGURE 6. Effect of the N-stearated ßc 450–465 peptide on the coimmunoprecipitation of Lyn or Jak2 and ßc. TF-1 cells were incubated in the presence or absence of the N-stearated ßc 450–465 peptide for 2 h. After lysing the cells, the cell lysates were immunoprecipitated with anti-Lyn or anti-Jak2 Ab. Western blotting with anti-ßc Ab revealed that coimmunoprecipitation of Lyn and ßc, but not Jak2 and ßc, was inhibited by the peptide, indicating the specific binding of the peptide to Lyn kinase in situ (n = 3).

 
Mapping the ßc binding site of Lyn kinase

The structural characteristic of Lyn kinase includes the presence of SH2 and SH3 domains (21). Lyn also has an N-terminal unique domain that has been shown to bind to Ig{alpha} in B cells (22) and Fc{epsilon}RIß in mast cells (21). The N terminus is myristoylated, which helps its juxtamembranous localization. To determine which domain of Lyn is responsible for the physical association with the ßc, we performed binding experiments using several Lyn-truncated GST fusion proteins. The GST-Lyn[1–243], GST-Lyn[1–61], and GST-Lyn[1–119] proteins possess the full-length Lyn without the catalytic domain, the Lyn-unique domain, and the Lyn-unique and SH3 domains, respectively. The GST-Lyn[1–243] protein was used as a positive control because the catalytic domain does not participate in the binding. We performed in vitro binding experiments of these fusion proteins with the ßc 462–481 peptide. We found that the GST-Lyn[1–61] and, to a lesser extent, GST-Lyn[1–119], bound to the ßc 462–481 peptide (Fig. 7GoA). In contrast, they did not bind to the mutated ßc 462–481 peptide with the P->A substitution (Fig. 7GoB). The data strongly suggest that Lyn is physically associated with the ßc receptor through its unique domain, although we cannot completely rule out the contribution of SH3 domain. We also used a Lyn SH2 domain containing protein, GST-Lyn[131–243], in the binding experiment. Unfortunately, this fusion protein bound not only the ßc 462–481 peptide, but also the mutated peptide, indicating that the interaction between GST-Lyn[131–243] and the ßc 462–481 peptide was nonspecific (data not shown).



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  		FIGURE 7. The binding of the ßc 462–481 peptide (A) and the mutated ßc 462–481 peptide (B) with P->A substitution to the Lyn-truncated GST fusion proteins. The GST-Lyn proteins or GST were precleared with avidin-agarose and then incubated with the ßc 462–481 peptide (50 µM) or the mutated ßc 462–481 peptide (50 µM). After incubation for 4 h, the avidin-agarose conjugates were added and the bound proteins were separated. The proteins were electrophoresed and Western blotted with anti-GST Ab. The GST-Lyn[1–243], GST-Lyn[1–61], and GST-Lyn[1–119] proteins possess the full-length Lyn without catalytic domain, the Lyn-unique domain, and the Lyn-unique and SH3 domains, respectively. Results are representative of one of three experiments. A, The GST-Lyn[1–243] protein was used as a positive control. The GST-Lyn[1–61] and, to a lesser extent, GST-Lyn[1–119] protein bound to the ßc 462–481 peptide. B, In contrast, none of them bound to the mutated ßc 462–481 peptide.

 
To further investigate whether Lyn binds to the ßc 462–481 peptide through the SH2 domain, we undertook another approach. The SH2-containing phosphatase-2 (SHP-2) is a tyrosine phosphatase, which has two SH2 domains and is known to bind to phosphorylated tyrosine residues on ßc through its SH2 domain. We studied the binding of SHP-2 to the ßc 462–481 peptide. We also used another peptide, ßc 605–624 (pY612), in which tyrosine 612 was phosphorylated. As anticipated, the ßc 605–624 (pY612) peptide, but not the ßc 462–481 peptide, bound SHP-2 (Fig. 8Go). The right lane containing the unprocessed cell lysate shows the position of SHP-2. The lack of binding to SHP-2 suggests that the physical association of the ßc 462–481 peptide to Lyn is unlikely to occur via the SH2 domain. One of our Lyn-binding peptides, the ßc 450–465 peptide, has two tyrosine residues. We were concerned that the ßc 450–465 peptide underwent phosphorylation during in vitro processing and bound to Lyn via the SH2 domain. However, in a previously published paper, we demonstrated that the ßc 450–465 peptide did not bind SHP-2 (13).



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  		FIGURE 8. The binding of the ßc 462–481 and the ßc 605–624 (pY612) to SHP-2. Human blood leukocytes were lysed and incubated with biotinylated ßc 462–481 (50 µM) or ßc 605–624 (pY612) (5 and 50 µM). Following precipitation with avidin-agarose, the eluates were electrophoresed and Western blotted with anti-SHP-2 Ab. The right lane containing the unprocessed cell lysate shows the position of the SHP-2. The result shows that the ßc 605–624 (pY612), but not the ßc 462–481, bound SHP-2 (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that the membrane-proximal 450–517 residues of the IL-5R ßc subunit are important for binding to Lyn, Jak2, and Fes kinases (9). Based on this information, we attempted to identify the exact Lyn binding site of the ßc using overlapping and/or sequential peptides derived from this region. In the present study, we narrowed down the Lyn binding site to a nonapeptide encompassing the residues ßc 457–465. We have shown that the proline residues from this region have a critical role in Lyn binding. Furthermore, we have demonstrated that a cell-permeable Lyn-binding peptide specifically blocks the binding of Lyn, but not Jak2 kinase to the ßc receptor in situ. Lyn kinase has an N-terminal unique domain, followed by SH3, SH2, and the catalytic domain. We present evidence that the unique domain of Lyn kinase mainly contributes to the physical association with the ßc receptor. This is the first identification of the molecular interaction site of ßc receptor with Lyn kinase.

We focused on Lyn kinase for the following reasons. Lyn kinase is associated with several receptors, including ßc (4, 6, 9), Fc{epsilon}RIß (14, 15), gp130 (16), Ig{alpha} (22), Fc{alpha}R (24), G-CSFR (25), CD14 (26), CD19 (27), and CD22 (28). The hematopoietins (IL-3, IL-5, G-CSF, and GM-CSF) are important for myeloid cell growth and differentiation. The Fc{epsilon}RIß is important for mast cell/basophil activation. Ig{alpha}, CD19, and CD22 are involved in B cell activation. The results from Lyn knockout mice suggest that Lyn plays a critical role in activation of mast cells (10). Lyn-/- B cells exhibited hyperresponsiveness to anti-IgM-stimulated proliferation (29, 30). Interestingly, however, Nishizumi et al. have shown that the Ca2+ mobilization, but not degranulation, is impaired in deficient mice (11). We have demonstrated that Lyn is important for eosinophil survival, but not for eosinophil degranulation or up-regulation of adhesion molecules (12). These results indicate that Lyn is involved in specific cellular functions of B cells, mast cells, and eosinophils. For this reason, it is important to understand the molecular basis of the interaction of Lyn kinase to receptors. The goal of this study was to map the Lyn binding site of the ßc subunit of IL-3/GM-CSF/IL-5 receptors.

Lyn has an N-terminal unique domain, followed by a SH3 domain, SH2 domain, and the tyrosine kinase domain. Thus, Lyn can interact with receptors and other signaling molecules via three different sites: unique domain, SH3 domain, and SH2 domain. The SH2 domain binds to tyrosine-phosphorylated residues. Three of our four Lyn-binding peptides do not have any tyrosine residues in their sequence, indicating that SH2 domain is not involved in Lyn binding. Tyrosine residues of the ßc receptor undergo phosphorylation only after receptor activation. Since Lyn kinase is associated with the ßc receptor in unstimulated cells, it is unlikely that this association is SH2 domain dependent. We have examined the binding of our peptides to SHP-2, which has two SH2 domains. To this goal we have selected the ßc 450–465 peptide, which has two tyrosine residues, and the ßc 462–481 peptide, which does not have any tyrosine residues in its sequence. Neither peptides bind to SH2, suggesting that the Lyn SH2 domain is not involved in binding to ßc receptor.

It has been demonstrated that the N-terminal 27 amino acid residues of Lyn are required for binding to the Ig{alpha}-chain Ag receptor homology 1 (ARH1) of the B cell Ag receptor (22). A direct interaction of Lyn with Fc{epsilon}RIß receptor through its unique domain has also been detected using the two-hybrid system (23). We tested the molecular interaction of the ßc 462–481 peptide with Lyn-truncated GST fusion proteins, and found that the peptide strongly bound to the unique domain of Lyn. The binding to GST-Lyn[1–119] protein, which comprises both the unique domain and SH3 domain, was weaker. Since we did not use a SH3 domain on its own, our study does not completely rule out the possibility that Lyn interacts with the ßc receptor via the SH3 domain as well. Recent crystallographic studies of Hck, a Lyn-related src family member, have shed new light on possible physiologic function of the SH3 domain (31). The core ligand-binding surface of the SH3 domain recognizes the proline-rich linker region between the SH2 and catalytic domains and physically associates with it. This action consequently let the RT loop of the SH3 domain contact the small lobe of the catalytic domain, which stabilizes the inactive form of the kinase. This structural study suggests that the SH3 domain may not be available for association with other signaling molecules. Given the structural homology among the src family members, the same principle may apply to Lyn SH3 domain. In support of this, our study clearly indicates that the unique domain alone is sufficient to interact with ßc peptides.

Jak2 is another tyrosine kinase that is constitutively associated with ßc receptor. The binding of Jak2 kinase to receptors has been studied by Murakami et al. (17). For this purpose they initially have studied the gp130 subunit of the IL-6R. They have postulated that the so-called box 1 region is important for signaling via the gp130 receptor subunit. The proline-rich motif (PXP motif) in the box 1 region is conserved among many cytokine receptors, e.g., ßc receptor of IL-3/GM-CSF/IL-5, IL-2Rß, G-CSFR, and erythropoietin receptor. The mutation of the two proline residues in the PXP motif of gp130 results in complete loss of IL-6 signaling activity. The deletion of box 1 from the receptors for growth hormone and erythropoietin also abolishes their Jak2-binding property (32). In accordance with this observation, Watanabe et al. have reported that the box 1 region is essential for GM-CSF-dependent Jak2 activation (33). Since this region corresponds to ßc 458–465, we examined the binding of our peptides to Jak2. However, we did not observe any interactions between Jak2 and the Lyn-binding peptides. This observation does not necessarily contradict the previous findings that the PXP motif is important for Jak2 binding. In support, Ogata et al. have recently shown that a GST fusion protein with the cytoplasmic region (amino acid residues 456–544) of the ßc, which contains the box 1 motif, cannot bind Jak2 (34). These results indicate that the box 1 region may be essential, but not sufficient for Jak2 binding. Clearly, a region of ßc located C terminally of the residue 544 participates in Jak2 binding. Recently, the binding sites for Jak1 and Jak3 have been studied using a series of truncated IL-2Rß mutants (35). Although two point mutations in the box 1 region of IL-2Rß decrease the binding of both Jak1 and Jak3, regions distal to box 1 appear to play major roles in the recruitment of Jak kinases.

In summary, we have delineated the exact Lyn binding site of the ßc receptor. We have shown previously that Lyn is constitutively associated with ßc receptor and has a key role in initiating downstream signals in eosinophils. Excessive production of eosinophils and their subsequent invasion of the airways and other target organs are characteristic features of asthma and allergic diseases. Additionally, there is evidence that the eosinophil survival in these diseases is prolonged due to the action of IL-5 and GM-CSF. Lyn is known to be essential for the maintenance of eosinophil survival (6, 12). The identification of the Lyn binding site of ßc receptor may help design specific inhibitors of IL-5 signaling. In this study, we have modified the Lyn-binding peptide by N-stearation to enable cellular internalization. The N-stearated peptide blocks the association of ßc with Lyn, but not Jak2. Thus, this peptide has the potential to inhibit Lyn-dependent IL-5 activities on eosinophils and may be useful for treatment of asthma and allergic diseases.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (AI35713) and from the James W. McLaughlin Foundation. T.A. was supported by a McLaughlin Postdoctoral Fellowship. K.P. is the recipient of a McLaughlin Postdoctoral Fellowship and President’s Grant-In-Aid Award from the AAAA&I. Back

2 Address correspondence and reprint requests to Dr. Rafeul Alam, Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Medical Branch, Clinical Sciences Bldg. 409, Galveston, TX 77555-0762. E-mail address: Back

3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; ßc, common ß; G-CSF, granulocyte colony-stimulating factor; GST, glutathione S-transferase; Jak, Janus kinase; SH, Src homology; SHP, Src homology 2-containing phosphatase. Back

Received for publication August 3, 1998. Accepted for publication October 9, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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