A chemical approach to the identification of tensin-binding proteins.
Many protein-protein interactions are mediated by small modular domains, which recognize short peptide motifs in their partner proteins. However, for the great majority of these domains, the identity of their partner proteins remains unknown. In this work, a chemical/bioinformatics approach has been developed to identify phosphotyrosyl (pY) proteins that bind to tensin, a protein involved in the formation of actin cytoskeleton and signal transduction. A pY peptide library was chemically synthesized and screened against the Src homology 2 (SH2) domain of tensin to identify the peptide motifs that bind to the SH2 domain. Next, protein databases were searched for proteins containing the SH2 domain-binding peptide motifs. Finally, the potential tensin-binding proteins were confirmed (or disproved) by in vitro pull-down and coimmunoprecipitation assays. This procedure identified phosphoinositide-dependent kinase-1 and downstream of tyrosine kinase 2 as novel tensin-binding proteins. In addition, a cell-permeable pY peptide was designed as tensin SH2 domain inhibitor, which caused the disruption of actin filaments in NIH 3T3 cells. This method should be generally applicable to other modular domains that recognize small peptide motifs.
Wavreille
Anne-Sophie
AS
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Pei
Dehua
D
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D019295:Computational Biology; D019992:Databases as Topic; D047468:Immunoprecipitation; D051379:Mice; D008840:Microfilament Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D041681:NIH 3T3 Cells; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D053634:TYK2 Kinase / Q000378:metabolism*; D000070876:Tensins; D018909:src Homology Domains
D002352:Carrier Proteins; D008840:Microfilament Proteins; D000070876:Tensins; D053634:TYK2 Kinase; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases