Identification of preferred protein interactions by phage-display of the human Src homology-3 proteome.
We have determined the human genome to contain 296 different Src homology-3 (SH3) domains and cloned them into a phage-display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3-directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2-binding adaptor protein POSH2 and the ADAM15-binding sorting nexin family member SNX30.
Kärkkäinen
Satu
S
Institute of Medical Technology, University of Tampere and Tampere University Hospital, Biokatu 8, Tampere 33014, Finland.
Hiipakka
Marita
M
Wang
Jing-Huan
JH
Kleino
Iivari
I
Vähä-Jaakkola
Marika
M
Renkema
G Herma
GH
Liss
Michael
M
Wagner
Ralf
R
Saksela
Kalle
K
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D020543:Proteome; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D054462:p21-Activated Kinases; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D002352:Carrier Proteins; D015693:Gene Products, nef; D008024:Ligands; D008565:Membrane Proteins; D019151:Peptide Library; D011506:Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C498193:SH3RF1 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D051722:ADAM Proteins; C098660:ADAM15 protein, human