Association of Grb-2 and PI3K p85 with phosphotyrosile peptides derived from BTLA.
B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory receptor expressed by B and T cells. We previously identified two tyrosine-containing signaling motifs in the cytoplasmic domain of BTLA that interact with the SHP-1 and SHP-2 phosphatases. BTLA has a third conserved tyrosine-containing motif within the cytoplasmic domain, similar in sequence to a Grb-2 recruitment site. To identify specific interacting proteins that would be recruited to this motif, we carried out an unbiased screen by using synthetic peptides in active (e.g., phosphotyrosil-containing) or control (e.g., non-phosphorylated) forms as baits. Using mass spectrometry, we identified two specific interacting proteins, Grb-2 and the p85 subunit of PI3K. Further, we demonstrate that the interaction with Grb-2 is direct, whereas the recruitment of the p85 subunit by BTLA phosphotyrosile-containing peptides may be indirect via its association with Grb-2. These findings may provide biochemical basis for previously unexplained actions of BTLA.
Gavrieli
Maya
M
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Murphy
Kenneth M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C482586:BTLA protein, human; D051380:GRB2 Adaptor Protein; D009842:Oligopeptides; D021122:Protein Subunits; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases