Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins.
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.
Mesh Terms:
Adaptor Proteins, Vesicular Transport, Adipose Tissue, Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Calcium, Calorimetry, Cricetinae, Crystallography, X-Ray, Electrons, Immunoblotting, Immunoprecipitation, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphorylation, Phosphotyrosine, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Signal Transduction, Thermodynamics, Transfection, Tyrosine
Adaptor Proteins, Vesicular Transport, Adipose Tissue, Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Calcium, Calorimetry, Cricetinae, Crystallography, X-Ray, Electrons, Immunoblotting, Immunoprecipitation, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphorylation, Phosphotyrosine, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Signal Transduction, Thermodynamics, Transfection, Tyrosine
J. Biol. Chem.
Date: May. 13, 2005
PubMed ID: 15737992
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