Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.
The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.
van der Geer
P
P
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON Canada.
Wiley
S
S
Gish
G D
GD
Lai
V K
VK
Stephens
R
R
White
M F
MF
Kaplan
D
D
Pawson
T
T
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D008024:Ligands; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000952:Antigens, Polyomavirus Transforming; D008024:Ligands; D010748:Phosphopeptides; D011506:Proteins; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D011972:Receptor, Insulin