Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening.
Target-assisted iterative screening applied to random peptide libraries unveiled a novel and atypical recognition consensus shared by CIN85/SETA/Ruk SH3 domains, PX(P/A)XXR. Confirmed by mutagenesis and in vitro binding experiments, the novel consensus allowed for the accurate mapping of CIN85 SH3 binding sites within known CIN85 interactors, c-Cbl, BLNK, Cbl-b, AIP1/Alix, ... SB1, and CD2 proteins, as well as the prediction of CIN85 novel-interacting partners in protein databases. Synaptojanin 1, PAK2, ZO-2, and TAFII70, which contain CIN85 SH3 recognition consensus sites, were selectively precipitated from mouse brain lysates by CIN85 SH3 domains in glutathione S-transferase pull-down experiments. A direct interaction of synaptojanin 1 and PAK2 with CIN85 SH3 domains was confirmed by Far Western blotting.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Alanine, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Biochemistry, Blotting, Western, Brain, Carrier Proteins, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Glutathione Transferase, Mice, Molecular Sequence Data, Mutagenesis, Mutagenesis, Site-Directed, Neoplasm Proteins, Nerve Tissue Proteins, Peptide Library, Phosphoric Monoester Hydrolases, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, src Homology Domains
Adaptor Proteins, Signal Transducing, Alanine, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Biochemistry, Blotting, Western, Brain, Carrier Proteins, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Glutathione Transferase, Mice, Molecular Sequence Data, Mutagenesis, Mutagenesis, Site-Directed, Neoplasm Proteins, Nerve Tissue Proteins, Peptide Library, Phosphoric Monoester Hydrolases, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, src Homology Domains
J. Biol. Chem.
Date: Sep. 05, 2003
PubMed ID: 12829691
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