Binding affinities of tyrosine-phosphorylated peptides to the COOH-terminal SH2 and NH2-terminal phosphotyrosine binding domains of Shc.
The adaptor protein Shc has been implicated in Ras signaling via association with many tyrosine-phosphorylated receptors, including growth factor receptors, antigen receptors on T and B cells, and cytokine receptors. Shc could interact with the activated receptors through the carboxyl-terminal Src homology 2 (SH2) domain or the structurally unrelated amino-terminal phosphotyrosine binding (PTB) domain. Using NMR and surface plasmon resonance techniques, we have measured the binding affinities of the SH2 and the PTB domains of Shc to a series of phosphotyrosine-containing peptides derived from known Shc binding sites. Tyrosine-phosphorylated peptides derived from Trk (pY490), polyoma virus middle T-antigen (pY250), ErbB3 (pY1309), and epidermal growth factor receptor (pY1086, pY1148, and pY1114) that contain NPXpY sequences bind preferentially to the PTB domain of Shc with Kd values of 0.02-5.3 microM. The binding affinities of these peptides to the Shc SH2 domain were in the range of 220-1290 microM. In contrast, tyrosine-phosphorylated peptides from epidermal growth factor receptor (pY992, pY1173) and the zeta chain of the T-cell receptor bind preferentially to the SH2 domain (Kd = 50-130 microM) versus the PTB domain (Kd > 680 microM). From these studies, the relative contribution of the individual domains of Shc for binding to the phosphotyrosine-containing portions of these proteins was determined. In addition, our data indicate that the high affinity binding of the PTB domain to the NPXpY-containing peptides results from a very high association rate and a rapid dissociation rate, which is similar to previous results observed for the SH2-phosphopeptide complexes.
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA.
M M
MM
Zhou
J E
JE
Harlan
W S
WS
Wade
S
S
Crosby
K S
KS
Ravichandran
S J
SJ
Burakoff
S W
SW
Fesik
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine