title: Adaptor protein Lnk associates with Tyr(568) in c-Kit.
abstract: 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 Tyr(568) 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.
authors.affiliation: Division of Hematology/Oncology, Cedars-Sinai Medical Center and UCLA School of Medicine, 8700 Beverly Boulevard, Los Angeles 90048, CA, USA. sgueller@gmail.com
authors.forename: Saskia
authors.initials: S
authors.lastname: Gueller
authors.forename: Sigal
authors.initials: S
authors.lastname: Gery
authors.forename: Verena
authors.initials: V
authors.lastname: Nowak
authors.forename: Liqin
authors.initials: L
authors.lastname: Liu
authors.forename: Hubert
authors.initials: H
authors.lastname: Serve
authors.forename: H Phillip
authors.initials: HP
authors.lastname: Koeffler
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C490778:SH2B3 protein, human; D014443:Tyrosine; D019009:Proto-Oncogene Proteins c-kit