Interaction of phosphorylated FcepsilonRIgamma immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics.
To examine the characteristics of the interaction of the FcepsilonRIgamma ITAM with the SH2 domains of p72(syk), the binding of an 125I-labeled dual phosphorylated FcepsilonRIgamma ITAM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72(syk) SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcepsilonRIgamma ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.
Chen
T
T
Department of Inflammation and Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110, USA.
Repetto
B
B
Chizzonite
R
R
Pullar
C
C
Burghardt
C
C
Dharm
E
E
Zhao
Z
Z
Carroll
R
R
Nunes
P
P
Basu
M
M
Danho
W
W
Visnick
M
M
Kochan
J
J
Waugh
D
D
Gilfillan
A M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D017931:DNA Primers; D004792:Enzyme Precursors / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008407:Mast Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017455:Receptors, IgE / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D000072377:Syk Kinase; D018909:src Homology Domains
D017931:DNA Primers; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D017455:Receptors, IgE; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase