Identification of residues within the SHC phosphotyrosine binding/phosphotyrosine interaction domain crucial for phosphopeptide interaction.
Shc is an Src homology 2 (SH2) domain protein thought to be an important component of the signaling pathway leading from cell surface receptors to Ras. A new phosphotyrosine interaction (PI) domain (also known as the phosphotyrosine binding (PTB) domain) has been described in the amino terminus of Shc. The Shc PI domain binding specificity is dependent on residues lying amino-terminal to the phosphotyrosine rather than carboxyl-terminal as is seen with SH2 domains. We randomly mutagenized the Shc PTB/PI domain in an effort to identify residues in the domain crucial for interaction with phosphotyrosine-containing peptides. We then screened the mutants for binding to the tyrosine-phosphorylated carboxyl-terminal tail of the epidermal growth factor (EGF) receptor. Most striking were mutations that altered a phenylalanine residue in block 4 of the domain severely impairing PI domain function. This phenylalanine residue is conserved in all but one subfamily of PI domains that have been identified to date. Reconstitution of this phenylalanine mutation into full-length Shc created a protein unable to interact with the EGF receptor in living cells.
Yajnik
V
V
Department of Pharmacology, New York University Medical Center, New York 10016, USA.
Blaikie
P
P
Bork
P
P
Margolis
B
B
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D066246:ErbB Receptors / Q000737:chemistry; D051379:Mice; D016296:Mutagenesis; D010748:Phosphopeptides / Q000737:chemistry*; D019000:Phosphotyrosine / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013329:Structure-Activity Relationship; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D010748:Phosphopeptides; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D066246:ErbB Receptors