Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting.
A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 x 10(5) individual peptides of nine amino acids in length (EPX6Y*X19X7X19X7X6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.
Müller
K
K
Sandoz Pharma Ltd., Preclinical Research, CH-4002 Basel, Switzerland.
Gombert
F O
FO
Manning
U
U
Grossmüller
F
F
Graff
P
P
Zaegel
H
H
Zuber
J F
JF
Freuler
F
F
Tschopp
C
C
Baumann
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002461:Cell Line, Transformed; D018076:DNA, Complementary; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000072377:Syk Kinase; D018909:src Homology Domains
D018076:DNA, Complementary; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D016650:Fluorescein-5-isothiocyanate