A protein-domain microarray identifies novel protein-protein interactions.
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.
The University of Texas M.D. Anderson Cancer Center, Science Park - Research Division, P.O. Box 389, Smithville, TX 78957, USA.
Alexsandra
A
Espejo
Jocelyn
J
Côté
Andrzej
A
Bednarek
Stephane
S
Richard
Mark T
MT
Bedford
D000595:Amino Acid Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase