Hu Junjie, Hubbard Stevan R
Structural Biology Program, Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, New York 10016, USA.
J Biol Chem. 2005 May 13;280(19):18943-9. doi: 10.1074/jbc.M414157200. Epub 2005 Feb 28.
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.
Cbl衔接蛋白通常通过将活化的蛋白酪氨酸激酶和其他信号蛋白与泛素化机制偶联，使其被蛋白酶体降解，从而发挥下调这些蛋白的功能。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，该结构域由一个四螺旋束、一个EF手型钙结合结构域和一个非常规的Src同源2结构域组成。先前推导的Cbl TKB结构域识别磷酸酪氨酸的共有序列为NXpY(S/T)XXP（X表示残基偏好性较低），其中特异性主要由磷酸酪氨酸C端的残基决定。在脂肪细胞中，Cbl通过衔接蛋白APS被胰岛素受体招募并磷酸化。APS在其C端酪氨酸残基上被胰岛素受体磷酸化，该残基随后作为Cbl TKB结构域的结合位点。通过X射线晶体学、定点诱变和量热研究，我们对Cbl TKB结构域与APS中Cbl招募位点之间的相互作用进行了表征，APS中的该位点包含一个序列基序RA(V/I)XNQpY(S/T)，在相关衔接蛋白SH2-B和Lnk中保守。这些研究揭示了磷酸肽与Cbl TKB结构域相互作用的一种新模式，其中磷酸酪氨酸远端的N端残基直接与TKB结构域四螺旋束的残基接触。
