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.
Kurakin
Alexei V
AV
Buck Institute for Age Research, Novato, California 94945, USA. akourakine@buckinstitute.org
Wu
Susan
S
Bredesen
Dale E
DE
D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C433810:SH3KBP1 protein, human; C498782:Sh3kbp1 protein, mouse; D005982:Glutathione Transferase; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D000409:Alanine