A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.
BACKGROUND
Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown.


RESULTS
We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine.


CONCLUSIONS
These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.
van der Geer
P
P
Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Wiley
S
S
Lai
V K
VK
Olivier
J P
JP
Gish
G D
GD
Stephens
R
R
Kaplan
D
D
Shoelson
S
S
Pawson
T
T
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D017124:Conserved Sequence; D066246:ErbB Receptors / Q000378:metabolism; D016391:Genes, src; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011506:Proteins / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D010748:Phosphopeptides; D011506:Proteins; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D066246:ErbB Receptors