Adaptor protein Lnk associates with Y568 in c-Kit
Résumé
The adaptor protein Lnk is expressed in hematopoietic cells and plays a critical role in hematopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in hematopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including a Src homology 2 (SH2) domain which binds phospho-tyrosines in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors (i.e. Mpl, EpoR, 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 hematopoiesis in response to exposure to cytokines (i.e. erythropoietin in anemic 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-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 the Y568 and was competed by peptides with a phospho-tyrosine in 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. 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 hematopoietic cells more sensitive to growth factors.
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