Novel Insights into the Mechanisms of CIN85 SH3 Domains Binding to Cbl Proteins: Solution-Based Investigations and In Vivo Implications Abdessamad Ababou 1 , Mark Pfuhl 2 and John E. Ladbury 1 ⁎ 1 Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK 2 Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK Received 10 October 2008; received in revised form 20 February 2009; accepted 24 February 2009 Available online 4 March 2009 CIN85 is a multifunctional protein that plays key roles in endocytic down-regulation of receptor tyrosine kinases, apoptosis, cell adhesion, and cytoskeleton rearrangement. Its three SH3 domains (CIN85A, CIN85B, and CIN85C) allow it to recruit multiple binding partners. To understand the manifold interactions of CIN85, we present a detailed high-resolution solution structural study of CIN85A and CIN85B binding to proline–arginine peptides derived from the cognate ligands Cbl and Cbl-b. We report the structure of CIN85B and provide evidence that both CIN85A and CIN85B, in isolation or when linked, form heterodimeric complexes with the peptides. We report unusual curved chemical shift changes for several residues of CIN85A when titrated with Cbl-b peptide, indicating the existence of more than one complex form. Here we demonstrate that CIN85A and CIN85B use different mechanisms for peptide binding. © 2009 Elsevier Ltd. All rights reserved. Edited by M. F. Summers Keywords: ubiquitination; NMR spectroscopy; isothermal titration calorimetry; chemical shifts Introduction The 85-kDa human Cbl-interacting protein CIN85, also known as SETA, Ruk, or SH3KBP1, is an important adaptor protein. 1 It is well known to play a key role in down-regulation of receptor tyrosine kinases and to facilitate endocytosis by interacting with endophilin-associated Cbl pro- teins. 2,3 However, CIN85 has also been implicated in other cellular processes such as apoptosis, via association with the p85 subunit of PI3K and AIP1/Alix, 4,5 as well as adhesion and cytoskeletal rearrangement, via interaction with p130Cas, cor- tactin, and ASAP1. 6–8 CIN85 is involved in additional important physiological processes such as regulation of cellular stress response via interaction with MEKK4, 9 cross-linking of F-actin into bundles, 10 and several others. 1,11–13 In addi- tion, CIN85 is implicated in infectious diseases via interaction with infected cell protein 0 from herpes simplex virus, 14 development of glomerulosclerosis in a diseased mouse model, 15 and breast cancer cell invasion via interaction with AMAP1. 16 CIN85 interacts with binding partners through its three SH3 domains (via atypical proline-rich motifs based on the sequence PXXXPR, where X is any amino acid), a proline-rich region, and a serine-rich region 10,17 (Fig. 1a). The importance of the CIN85 SH3 domains in clustering Cbl proteins has also been reported. 17 Although there are several iso- forms of CIN85, little is known about their roles in promoting or rescuing activity or in competing with full-length functions 5,18 (Fig. 1a). Nonetheless, the full-length protein and the isoform lacking the first SH3 domain show distinct functions. 19 CIN85 lacking the first two SH3 domains competes with the intact protein for binding to the p85α subunit of PI3K. 5 *Corresponding author. Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail address: jeladbury@mdanderson.org. Present address: A. Ababou, Department of Chemistry & Biological Sciences, University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK. Abbreviations used: ITC, isothermal titration calorimetry; CS, chemical shift; NOESY, nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single quantum coherence; 1D, one-dimensional. doi:10.1016/j.jmb.2009.02.061 J. Mol. Biol. (2009) 387, 1120–1136 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.