The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr568 and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.
The receptor for SCF (stem cell factor), c-Kit, belongs to the class III family of receptor tyrosine kinases that also includes PDGFR (platelet-derived growth factor receptor) and the macrophage CSF1R (colony-stimulating factor 1 receptor) [1,2]. These receptors are structurally characterized by an extracellular region consisting of five immunoglobulin-like domains, a transmembrane, JXM (juxtamembrane) and an intracellular tyrosine kinase domain. The latter is split into a proximal (Kin1) and a distal (Kin2) region separated by an insert. In general, c-Kit is expressed on haemopoietic stem cells and their progenitor cells, and expression is lost during further differentiation. It plays an important role in self-renewal, survival and growth of haemopoietic stem cells. Therefore homozygous c-Kit-knockout mice are non-viable owing to anaemia, and heterozygous deletional mice have reduced c-Kit tyrosine kinase activity causing anaemia and reduced fertility .
Binding of SCF to c-Kit results in receptor dimerization and activation of the tyrosine kinase, which subsequently causes autophosphorylation of specific tyrosine residues. These phosphotyrosine residues can be recognized and bound by signal transduction molecules containing SH2 (Src homology 2) domains. Therefore Grb (growth-factor-receptor-bound protein) 2 and 7, APS [adapter protein with PH (pleckstrin homology) and SH2 domains], Lnk, PI3K (phosphoinositide 3-kinase), PLCγ (phospholipase Cγ) and other proteins can associate with activated c-Kit .
Lnk is an adaptor protein expressed in haemopoietic tissues and has critical functions in haemopoiesis [5–10]. Together with APS and SH2-B, Lnk shares several structural motifs including a SH2 domain, a PH domain and a dimerization domain. In response to ligand-mediated autophosphorylation of tyrosine residues within EpoR (erythropoietin receptor) , thrombopoietin receptor (Mpl)  or SCF receptor (c-Kit) [6,8], Lnk associates with the receptors through its SH2 domain and subsequently inhibits their downstream signalling [13–15]. The SH2 domain is essential for the inhibitory activity of Lnk [6,11,12]. Proliferation and differentiation of haemopoietic precursors is enhanced by decreased expression of Lnk and is inhibited by increased levels of Lnk [6,8]. Therefore inhibition of Lnk binding to cytokine receptors makes haemopoietic progenitor cells more sensitive to the growth-enhancing factors. The binding site of Lnk in c-Kit has not yet been determined. We wished to determine this site of interaction because we want to develop an assay to screen for agents that prevent binding of Lnk to cytokine receptors and thereby enhance the effect of endogenous or exogenous cytokines.
Cell culture and transfections
HEK-293T (human embryonic kidney) cells were grown in DMEM (Dulbecco's modified Eagle's medium) with 10% FBS (fetal bovine serum) and transfected using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. At 48 h after transfection, cells were lysed in lysis buffer containing 0.5% Nonidet P40, 50 mM Tris/HCl (pH 7.5), 150 mM NaCl and Complete™ protease inhibitor cocktail (Roche Applied Science).
V5-tagged human Lnk cDNA was cloned into the pcDNA3.1 vector. The R392E Lnk point mutation was generated by PCR and confirmed by sequencing. DNA corresponding to the JXM (amino acids 544–577), Kin1 (amino acids 578–685), KI (kinase insert; amino acids 686–762), Kin2 (amino acids 775–845) and CT (C-terminal tail; amino acids 926–976) domains of c-Kit cloned into pGEX-4T-1 were kindly provided by Dr Johan Lennartsson (Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden). The Y568F and Y570F point mutations were generated by site-directed mutagenesis of the JXM domain, and the sequences confirmed by nucleotide sequencing. The expression vector (pDSRα22) for wild-type full-length c-Kit was kindly provided by Amgen. The Y568F point mutation was generated using the QuikChange® mutagenesis kit (Stratagene) followed by nucleotide sequencing.
GST (glutathione transferase)-fusion proteins
The pGEX-4T-1 constructs were transformed into TKX1 competent Escherichia coli cells (Stratagene) for production of GST-fusion proteins with phosphorylated tyrosine residues and into E. coli DH5α for generation of unphosphorylated GST-fusion proteins. Transformation, accumulation of the fusion proteins and induction of the tyrosine kinase were performed according to the manufacturer's instructions. Briefly, after growing either TKX1 or DH5α bacteria overnight, production of the GST-fusion proteins was induced using 0.1 mM IPTG (isopropyl β-D-thiogalactoside) (Invitrogen) for 2.5 h. Thereafter, only TKX1 bacteria were resuspended in tryptophan-free medium containing 10 μg/ml IAA (indoleacrylic acid) (Sigma–Aldrich) to activate the tyrosine kinase and were incubated for another 2 h. Harvesting and purification of phosphorylated and unphosphorylated GST-fusion proteins were carried out as described in the ‘GST Gene Fusion System’ manual (GE Healthcare). Cells were sedimented and disrupted by sonication, proteins were bound to gluthatione–Sepharose 4B (GE Healthcare) and were stored in PBS containing 15% glycerol at −80 °C until their use for pull-down experiments.
In vitro translation
IVT (in vitro translated) Lnk was synthesized from the pcDNA3.1-Lnk expression vector using the TNT® T7 coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. Generation of proteins was carried out in reticulocyte lysate using the one-step transcription/translation system. Expression levels of IVT Lnk were determined by Western blot.
GST pull-down assays were carried out with equivalent amounts of GST and GST-fusion proteins immobilized on glutathione–Sepharose beads in lysis buffer. After a 16 h incubation at 4 °C with whole HEK-293T cell lysates or IVT Lnk respectively, precipitated proteins were washed with PBS, eluted with SDS sample buffer and resolved by SDS/PAGE (4–15% gels). After transfer on to PVDF membranes, the Lnk protein was detected with anti-V5-tag antibodies (Applied Biological Materials), phosphorylation of the GST-fusion proteins was detected with anti-phosphotyrosine antibodies (Santa Cruz Biotechnology), and equal loading was confirmed either with anti-GST antibodies (Santa Cruz Biotechnology), Ponceau staining or Coomassie Blue staining of the gel. Phosphorylation of TKX1-derived GST-fusion proteins was verified by Western blot analysis using anti-phosphotyrosine antibodies.
For co-immunoprecipitation of full-length c-Kit and Lnk, HEK-293T cells were co-transfected with wild-type or Y568F mutant c-Kit and wild-type Lnk cDNA. Before lysis, cells were serum-starved overnight and treated with 50 ng/ml human SCF (Peprotech) for 30 min. Then, 500 μg of protein was precipitated using 1 μg of anti-V5-tag (Invitrogen) or anti-c-Kit (Santa Cruz Biotechnology) antibodies and 20 μl of Protein A/G Plus–agarose (Santa Cruz Biotechnology) at 4 °C for 16 h, then washed with PBS, eluted with SDS sample buffer and resolved by SDS/PAGE (4–15% gels). Precipitated proteins were detected with anti-c-Kit or anti-V5-tag antibodies.
Synthetic peptides corresponding to the tyrosine-containing part of the JXM domain of c-Kit were purchased from Quality Controlled Biochemicals including different phosphotyrosine (pY) residues: GKGKGKEEINGNNYVYIDPTQL-OH (Tyr568/Tyr570), GKGKGKEEINGNN[pY]VYIDPTQL-OH (pTyr568), GKGKGKEEINGNNYV[pY]IDPTQL-OH (pTyr570) and GKG- KGKEEINGNN[pY]V[pY]IDPTQL-OH (pTyr568/pTyr570). Competition pull-down assays were carried out in lysis buffer using a 10- or 100-fold molar excess of the peptides compared with the amount of GST–JXM fusion protein and 5 μg of protein from HEK-293T cells overexpressing Lnk. The samples were incubated at 4 °C for 2.5 h and then washed and processed as described for the pull-down assays.
Lnk binds to the phosphorylated JXM domain of c-Kit
Phosphorylated GST-fusion proteins consisting of the five different domains of c-Kit were isolated from TKX1 bacteria, and expression and phosphorylation were confirmed by Western blotting (Figure 1A). Some of the GST-fusion proteins showed low expression, so that the amounts of GST input were adapted according to their expression to get equal loading. In pull-down experiments using the different c-Kit domains that were incubated with whole-cell lysates of HEK-293T cells overexpressing wild-type Lnk, we found that Lnk mainly bound to the JXM domain of c-Kit (Figure 1B). As expected, Lnk did not bind to either the unphosphorylated JXM fusion protein or the control GST without fusion product.
Lnk binds to the JXM domain of c-Kit
Mutation of Tyr568 abolishes binding of Lnk
The SH2 domain of various adaptor proteins recognize and bind to phosphorylated tyrosine residues of their binding partners. Therefore we tested whether Lnk binds to a tyrosine residue in c-Kit. The JXM domain of the receptor carries two tyrosine residues that are potential binding sites for Lnk: Tyr568 and Tyr570. Using site-directed mutagenesis, we created vectors containing the sequences for the JXM domain of c-Kit containing either the Y568F or the Y570F point mutation respectively. Pull-down experiments were carried out with wild-type or point-mutated GST-fused JXM domains and whole-cell lysates derived from HEK-293T cells overexpressing Lnk. We found that the Y570F mutation did not influence the binding of Lnk, but the Y568F mutation abolished binding of Lnk to the JXM domain (Figure 2A). We conclude that Lnk selectively binds to phosphorylated Tyr568 in the JXM domain of c-Kit. To confirm the specificity of binding of Lnk to Tyr568 of c-Kit, competition assays using peptides with different phosphorylation patterns as competitors for GST–JXM were performed. The peptides were utilized in a 10- or 100-fold molar excess over GST–JXM. As shown in Figure 2(B), binding of Lnk to GST–JXM was competed in a dose-dependent manner by peptides harbouring a phosphotyrosine residue at position 568 (pTyr568/pTyr570 and pTyr568), whereas the other peptides did not affect binding. Furthermore, co-immunoprecipitation assays were performed to prove the specific binding site in full-length c-Kit. Using anti-V5-tag or anti-c-Kit antibodies respectively, proteins from HEK-293T lysates overexpressing either wild-type or Y568F mutant c-Kit and V5-tagged wild-type Lnk were precipitated and analysed by SDS/PAGE. Figure 2(C) shows that SCF-induced phosphorylation of tyrosine residues in wild-type c-Kit-expressing cells led to binding of Lnk to the receptor. The Y568F mutation completely abolished the interaction, thus showing the importance of this tyrosine as a docking site for Lnk.
Phosphorylated Tyr568 is the binding site of Lnk in c-Kit
Binding of human Lnk to human c-Kit depends on its SH2 domain
Binding of murine Lnk to the murine c-Kit receptor is abolished by point mutation of Arg364 of Lnk . We tested whether the equivalent arginine residue (Arg392) in the conserved SH2 domain of human Lnk is responsible for its binding to human c-Kit. A point mutation R392E was inserted into the Lnk vector and was expressed in HEK-293T cells. Pull-down experiments were performed using GST–JXM of c-Kit and either wild-type or mutant Lnk lysates. As shown in Figure 3, R392E completely abolished binding of Lnk to c-Kit.
Binding of Lnk depends on Arg392 in its SH2 domain
Binding of Lnk to c-Kit JXM is direct
Studies have shown that Lnk binds to c-Kit , but it has not been elucidated whether the binding is direct or whether Lnk needs co-partners to interact with c-Kit. Therefore pull-down assays were carried out using IVT protein as a source of Lnk instead of whole-cell lysates. Similarly to Lnk from HEK-293T lysates, IVT Lnk was able to bind to GST–JXM. Y570F point mutated GST–JXM still bound Lnk, whereas the Y568F point mutation abolished binding (Figure 4). These data demonstrate that Lnk binds to c-Kit directly and independently of other binding partners.
Lnk binds to c-Kit directly without requirement of co-partners
In the present study, we have demonstrated for the first time that the adaptor protein Lnk binds to the JXM domain of c-Kit and specifically to Tyr568. These results are in agreement with data that showed that APS, a family member of Lnk, binds to the same tyrosine residue in c-Kit . In contrast with APS, Lnk binds to only one tyrosine residue and does not bind to the CT domain of c-Kit; whereas APS binds to both the JXM and the CT domain. Both adaptor proteins led to inhibition of downstream signalling of c-Kit upon stimulation with SCF. The JXM domain of c-Kit is known to have an autoinhibitory function. Transphosphorylation of Tyr568 and Tyr570 are the first steps that occur after ligandmediated dimerization of the receptor. As a result, the JXM segment no longer immobilizes the receptor in a static configuration, but allows conversion of the activation loop of c-Kit from a compact inactive form into an extended activated receptor. Subsequently, transphosphorylation of Tyr823 in the activation loop stabilizes the active form of the tyrosine kinase . SHPs (SH2-domain-containing phosphatases) 1 and 2 are known to bind to phosphorylated Tyr568 and Tyr570 and terminate the SCF signal by dephosphorylating the two tyrosine residues and thereby restoring the inactive conformation of the receptor . Thus the JXM domain plays a major role both in activating and inhibiting c-Kit, which makes its tyrosine residues very important regulating elements. This is also reflected by the fact that several signal transduction molecules influencing the activity of the receptor (i.e. Src kinases, Chk and Shc) are known to bind to the same domain. We showed that binding of Lnk to this site is independent of binding other proteins, but Lnk might still be an important factor that mediates binding of other regulating proteins to the receptor. APS has been shown to bind c-Cbl, a ubiquitin ligase which targets growth factor receptors for degradation in lysosomes . Therefore loss of binding of APS to PDGFRβ leads to decreased recruitment of c-Cbl and to reduced degradation of the receptor, followed by enhanced c-fos activation. In contrast, low levels of c-Cbl had no effect alone on levels of the receptor, but, in the presence of APS, enhanced recruitment of c-Cbl occurred, resulting in degradation of the receptor . The C-terminal part of Lnk contains a c-Cbl binding site similar to that of APS, so the same mechanism could lead to its inhibitory function, whereas c-Cbl can also bind directly to c-Kit . Nevertheless, further exploration is necessary to elucidate the interaction between Lnk and c-Kit.
Our data identifying the binding site of Lnk to c-Kit can help to develop an assay to screen for ‘small-inhibitor molecules’ to prevent the binding of these two proteins, and thus potentially make haemopoietic cells more sensitive to either endogenous or exogenous haemopoietic growth factors offering a novel approach to stimulate blood cell production.
This work was supported in part by the Deutsche Krebshilfe (to S. Gueller) and the Tower Cancer Research Foundation Fellowship (to S. Gueller).
adapter protein with pleckstrin homology and Src homology 2 domains
human embryonic kidney
in vitro translation
pleckstrin homology domain
platelet-derived growth factor receptor
stem cell factor
Src homology domain 2