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Figure 1.
Figure 1. Gads Binding Proteins Share an Amino Acid Motif
Similar to SLP-76(A) Refinement of the minimal Gads SH3 domain
binding motif in SLP-76. Peptides (15-mer), scanning through the
binding region of SLP-76, were synthesized and spotted on a
cellulose filter as previously described [9]. The filter was
blocked with 5% skim milk powder in TBS-T and probed with
full-length GST-Gads C-SH3 fusion protein (2 μg/ml for 3 hr).
Bound fusion protein was detected by blotting with monoclonal
anti-GST antibody.(B) Alignment of known and novel Gads SH3
domain binding proteins reveals a common binding motif. A 16-day
mouse embryo protein library (Novagen) was screened with
GST-Gads fusion protein (see Supplementary Material). Isolated
cDNAs were partially sequenced and identified through the
GenBank database. A search of the GenBank database for other
proteins with the consensus PxxxR-X-X-KP identified both Gab2
and BLNK.(C) UBPY, Gab2, and BLNK can associate with Gads. Cos1
cells were transfected with 2 μg of pCMV-UBPY or pPuro-BLNK-Myc
and harvested 24–48 hr later. Clarified cell lysate from
transfected Cos1 cells or from K562 cells was incubated with 2
μg of GST or GST-Gads bound to glutathione-sepharose beads for
1–2 hr at 4°C. Beads were washed with NP-40 lysis buffer,
and bound proteins were separated by SDS-PAGE and Western
blotted with anti-UBPY (a gift from Paolo Di Fiore and Guillio
Draetta), anti-myc (to detect myc-BLNK), or anti-Gab2 (Upstate
Biotechnology).(D) Confirmation of binding sites in UBPY, AMSH,
and BLNK. Peptides representing the predicted binding region of
each protein were synthesized and spotted onto a cellulose
filter as in (A). The filters were probed with Gads GST-C-SH3
fusion protein (2 μg/ml) and blotted with anti-GST monoclonal
antibody (Upstate Biotechnology).
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