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J. Biol. Chem. 278 (36): 34102-34109

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Atypical Recognition Consensus of CIN85/SETA/Ruk SH3 Domains Revealed by Target-assisted Iterative Screening*

Alexei V. Kurakin {ddagger}, Susan Wu, and Dale E. Bredesen

Buck Institute for Age Research, Novato, California 94945

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.

Received for publication May 20, 2003.

* This work was supported in part by National Institutes of Health Grants NS45093 and AG12282 (to D. E. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{ddagger} To whom correspondence should be addressed: Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945. Tel.: 415-209-22-84; Fax: 415-209-22-30; E-mail: akourakine{at}buckinstitute.org.

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