Protein domains mediate protein—protein interactions through binding to short peptide motifs in their corresponding ligands. These peptide recognition modules are critical for the assembly of multiprotein complexes. We have arrayed glutathione S-transferase (GST) fusion proteins, with a focus on protein interaction domains, on to nitrocellulose-coated glass slides to generate a protein-domain chip. Arrayed protein-interacting modules included WW (a domain with two conserved tryptophans), SH3 (Src homology 3), SH2, 14.3.3, FHA (forkhead-associated), PDZ (a domain originally identified in PSD-95, DLG and ZO-1 proteins), PH (pleckstrin homology) and FF (a domain with two conserved phenylalanines) domains. Here we demonstrate, using peptides, that the arrayed domains retain their binding integrity. Furthermore, we show that the protein-domain chip can ‘fish’ proteins out of a total cell lysate; these domain-bound proteins can then be detected on the chip with a specific antibody, thus producing an interaction map for a cellular protein of interest. Using this approach we have confirmed the domain-binding profile of the signalling molecule Sam68 (Src-associated during mitosis 68), and have identified a new binding profile for the core small nuclear ribonucleoprotein SmB′. This protein-domain chip not only identifies potential binding partners for proteins, but also promises to recognize qualitative differences in protein ligands (caused by post-translational modification), thus getting at the heart of signal transduction pathways.
- arginine methylation
- proline-rich motifs
Abbreviations used: FF domain, a domain with two conserved phenylalanines (F); FHA, forkhead-associated; GST, glutathione S-transferase; KH, ribonucleoprotein K homology; PBST, PBS containing 0.1% Tween 20; PDZ domain, a domain originally identified in PSD-95, DLG and ZO-1 proteins; PGM, proline/glycine/methionine; PH, pleckstrin homology; PTB, phosphotyrosine-binding; Sam68, Src-associated during mitosis 68; SH3, Src homology 3; SH2, Src homology 2; WBP, WW-domain-binding protein; WW domain, a domain with two conserved tryptophans (W).
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