Abstract
The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.
Highlights
The SH2B family of adaptor proteins, Src homology 2 (SH2)-B, APS, and LNK are key modulators of cellular signalling pathways
The LNK SH2 domain was co-crystallised with a 12-mer phosphopeptide corresponding to JAK2 pY813 and flanking residues
The crystal structure of this complex was solved to 1.9 Å resolution (Supplementary Table 1), revealing that the LNK SH2 domain adopts a typical SH2 domain fold comprising three central β-strands flanked by two α-helices
Summary
The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. The Lymphocyte adaptor protein (LNK1 ( SH2B3)) is a member of the SH2 domain containing adaptor family of proteins, which comprises APS (SH2B2) and SH2B (SH2B1)[2,3] and negatively regulates both EPO and TPO signalling[4,5] via its interaction with JAK26. Overexpression of LNK in haematopoietic progenitor cell lines restrains TPO-induced cellular proliferation, and overexpression of LNK in primary hematopoietic cells inhibits megakaryopoiesis[4]. These findings are recapitulated in vivo with LNK-deficient mice displaying increased numbers of megakaryocytes that have enhanced TPO sensitivity[4] as well as enhanced numbers of platelets, lymphocytes and erythroid cells[3,5]. Understanding how the LNK SH2 domain interacts with substrates may shed light on how negative regulation of various signalling molecules occurs, and why mutations in the LNK SH2 domain contribute to disease burden in patients
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