Plaza-Menacho Iván, Barnouin Karin, Barry Rachael, Borg Annabel, Orme Mariam, Chauhan Rakhee, Mouilleron Stephane, Martínez-Torres Rubén J, Meier Pascal, McDonald Neil Q
Structural Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Protein Analysis and Proteomics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Cell Rep. 2016 Dec 20;17(12):3319-3332. doi: 10.1016/j.celrep.2016.11.061.
Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.
尽管受体酪氨酸激酶具有高度同源的催化结构域，但它们表现出多种激活机制。这种多样性是通过细胞外配体结合部分与酪氨酸激酶结构域两侧高度可变的细胞内序列以及自磷酸化位点的特定模式偶联而产生的。在这里，我们表明近膜（JM）段通过Y687增强RET催化结构域的活性。这个磷酸化位点也是JM区域拯救否则缺乏酪氨酸的催化缺陷型RET激活环突变体所必需的。结构-功能分析确定了JM铰链、αC螺旋与一个非常规激活环丝氨酸磷酸化位点之间的相互作用，该位点与HRD基序结合并促进磷酸酪氨酸构象可及性和调节脊柱组装。我们证明这个磷酸化的S909来自内在的RET双特异性激酶活性，并表明果蝇中RET信号传导需要一个等效的丝氨酸。我们的发现揭示了RET激活和信号传导机制的双特异性和变构成分，对药物发现具有直接影响。
