Abstract
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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Alanine / chemistry
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Amino Acid Sequence
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Animals
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Base Sequence
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Binding Sites
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Biochemistry / methods
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Blotting, Western
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Brain / metabolism
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism
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Electrophoresis, Polyacrylamide Gel
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Enzyme-Linked Immunosorbent Assay
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Glutathione Transferase / metabolism
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Mice
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Molecular Sequence Data
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Mutagenesis
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Mutagenesis, Site-Directed
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Neoplasm Proteins / chemistry*
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Neoplasm Proteins / metabolism
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Nerve Tissue Proteins / chemistry*
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Nerve Tissue Proteins / metabolism
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Peptide Library
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Phosphoric Monoester Hydrolases / chemistry
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Precipitin Tests
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Protein Binding
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / metabolism
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src Homology Domains
Substances
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Adaptor Proteins, Signal Transducing
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Carrier Proteins
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Neoplasm Proteins
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Nerve Tissue Proteins
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Peptide Library
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Recombinant Fusion Proteins
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SH3KBP1 protein, human
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Sh3kbp1 protein, mouse
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Glutathione Transferase
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synaptojanin
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Phosphoric Monoester Hydrolases
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Alanine