SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.
src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-gamma and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosine-containing regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BIAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 x 10(7) to 40 x 10(7)/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.
Felder
S
S
Department of Pharmacology, New York University Medical Center, New York 10016.
Zhou
M
M
Hu
P
P
Ureña
J
J
Ullrich
A
A
Chaudhuri
M
M
White
M
M
Shoelson
S E
SE
Schlessinger
J
J
D000595:Amino Acid Sequence; D016391:Genes, src / Q000235:genetics; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017385:Sequence Homology; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases