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Figure 4.
Figure 4 EAT-2 binds exclusively to a phosphorylated peptide
(pY281) derived from the cytoplasmic tail of CD150. (A)
Fluorescence polarization analysis of the EAT-2 binding to a
phosphorylated pY281 peptide. Different concentrations of GST
-mouse EAT-2 (or GST -human SH2D1A) and an 11mer synthetic
peptide identical to amino acid residues 276 -287 of human CD150
(Sayos et al., 1998), tyrosine phosphorylated or not, were used.
Top panel: binding of GST -mouse EAT-2 to the pY281 (filled
triangles and continuous line) or the Y281 peptide (open squares
and dashed line). Bottom panel: binding of GST -human SH2D1A to
the pY281 (filled triangles and continuous line) or the Y281
peptide (open squares and dashed line). x-axis: protein
concentration (nM); y-axis: polarization units (mP). The table
summarizes the apparent dissociation constant (kD). (B) Hybrid
system analysis of the interaction between EAT-2 and the
cytoplasmic tail of CD150 in the presence or absence of fyn.
Dashed bars indicate the interaction between the EAT-2 (or
SH2D1A) full-length protein fused to a GAL4 DNA-binding domain
and the GAL4 DNA activation domain fused to the cytoplasmic tail
of the CD150 receptor. An empty pGAD424 vector was used as a
control (solid bars). The test was conducted in either the
presence or absence of fyn[420,531Y -F]. y-axis =  -galactosidase
(U/ml).
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