Multimeric and differential binding of CIN85/CD2AP with two atypical proline‐rich sequences from CD2 and Cbl‐b*

Abstract

The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways. Abbreviations Cbl, Casitas B-lineage lymphoma; CD2AP, CD2-associated protein; CD2, cluster of differentiation 2; CIN85, Cbl-interacting protein of 85 kDa; CMS, Cas ligand with multiple SH3 domains; SH3 domain, Src homology 3 domain.

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

“…Thus, deviations in the loops' positions found in SH3-A may prevent stable docking of the arginine. This is reminiscent of recent work showing how the structure of the n-Src loop of CIN85 SH3-A, but not of CD2AP SH3-A, is able to promote heterotrimer formation, that is, two SH3 domains clamping onto a single c-Cbl peptide, and thus giving rise to both class I and II ligand binding [43]. Other notable differences in coordinate positions include those of the C-terminal peptide region.…”

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)

“…Subsequently, solution data were inconsistent with heterotrimer formation; rather, CIN85 SH3-1 most likely supported either class I or II binding in independent heterodimers (59). Most recently, a detailed biophysical study of the solution state revealed important contrasts between CIN85 and CD2AP (60). CD2AP appeared only to form heterodimers with CD2 or CBL-b peptides.…”

“…P 1 (K/L) 2 PxPR 6 ), both of which have been proven to enable distinct partner peptides to bind in the reverse class I orientation under certain conditions (45). In addition, Arg in motif position Ϫ2, which could promote class I binding (58,60), is absent.…”

Septin 9 negatively regulates ubiquitin-dependent downregulation of epidermal growth factor receptor

“…Moreover, titration of the peptide induced changes in chemical shifts of amino acids implicated in binding to other PRsignature motifs (Fig. 5B) (Ceregido et al, 2013), indicating that both ligands compete for the same binding site. Accordingly, SEPT9-and Cbl-derived peptides occupied the same binding surface on SH3A domain of CIN85 (Fig.…”

“…Purified SH3A was dialyzed overnight into a buffer containing 50 mM cacodylate pH 7.4. A reference assignment of the resonances of the 1 H, 15 N-HSQC of the protein was kindly provided by others (Ceregido et al, 2013). Because the protein construct used here slightly differed from the one documented previously, we performed measurements to confirm the assignment of the protein resonances.…”