Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins
Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins
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DOI：
10.1074/jbc.m414157200
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发表时间：
2005-05-13
影响因子：
4.8
通讯作者：
Hubbard, SR
中科院分区：
文献类型：
--
作者：
Hu, JJ;Hubbard, SR
Cbl适配器蛋白通常通过将酪氨酸激酶和其他信号蛋白偶联到蛋白酶体降解的泛素化机制上，从而下调活化蛋白酪氨酸激酶和其他信号蛋白的功能。Cbl蛋白通过酪氨酸激酶结合（TKB）结构域与靶蛋白中特定的酪氨酸磷酸化序列结合，该结构域包括一个四螺旋束、一个EF-hand钙结合结构域和一个非常规的Src同源-2结构域。先前导出的Cbl TKB结构域识别磷酸酪氨酸的共识序列是NXpY(S/T)XXP （X表示较小的残基偏好），其中特异性主要由磷酸酪氨酸的残基c端赋予。Cbl通过适配蛋白APS被脂肪细胞中的胰岛素受体募集并磷酸化。APS被c端酪氨酸残基上的胰岛素受体磷酸化，然后作为Cbl TKB结构域的结合位点。利用x射线晶体学、定点诱变和量热研究，我们表征了APS中Cbl TKB结构域和Cbl募集位点之间的相互作用，其中包含一个序列基序RA(V/I)XNQpY(S/T)，该序列基序在相关的连接蛋白SH2-B和Lnk中保守。这些研究揭示了一种新的磷酸肽与Cbl TKB结构域相互作用的模式，其中远端磷酸酪氨酸的n端残基直接接触TKB结构域的四螺旋束残基。
The Cbl adapter proteins typically function to down-regulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the four-helix bundle of the TKB domain.