Identification of a Novel Proline-Arginine Motif Involved in CIN85-dependent Clustering of Cbl and Down-regulation of Epidermal Growth Factor Receptors

Katarzyna Kowanetz,Iwona Szymkiewicz,Kaisa Haglund,Marcin Kowanetz,Koraljka Husnjak,Jonathan D Taylor,Philippe Soubeyran,Ulla Engstrom,John E Ladbury,Ivan Dikic

doi:10.1074/jbc.m304541200

Abstract

CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.

Highlights

Lular signaling proteins with receptors, and phosphorylation of multiple substrates [1]
CIN85 binds to Cbl via its SH3 domains, and their association is enhanced by growth factor-induced tyrosine phosphorylation of Cbl [15, 17], whereas the proline-rich region of CIN85 constitutively interacts with endophilins [11, 12], regulatory components of clathrin-coated pits [18]
More detailed analysis indicated that mutation of proline 906 in Cbl-b peptide (PXXPAXPXPR) significantly reduced its ability to associate with CIN85, and the effect was even more prominent when proline 906 was mutated together with proline 910 in Cbl-b sequence (PXXPAXPXAR) but not when mutated together with proline 905 (PXXAAXPXPR) (Fig. 2B, right panel)

Summary

Introduction

Lular signaling proteins with receptors, and phosphorylation of multiple substrates [1]. The multidomain structure of CIN85 and its homologue CMS/CD2AP enables them to associate with various proteins including Cbl, Grb, p85 subunit of phosphatidylinositol 3-kinase, CD2 receptors, SETA-binding protein 1 (SB-1), SLP-65/BLNK, Alg2-interacting protein 1 (AIP1), and p130Cas [16] These interactions promote formation of CIN85-linked protein networks that are implicated in the control of RTK signaling, actin reorganization, T cell functions, kidney architecture, and apoptotic signals [16]. CIN85 binds to Cbl via its SH3 domains, and their association is enhanced by growth factor-induced tyrosine phosphorylation of Cbl [15, 17], whereas the proline-rich region of CIN85 constitutively interacts with endophilins [11, 12], regulatory components of clathrin-coated pits [18] Based on these features, CIN85 can rapidly recruit endophilins to complexes with activated receptors, controlling receptor inter-. CIN85 can function as an RTK scaffold molecule, like G proteincoupled receptor specific ␤-arrestins, controlling receptor endocytosis and degradation

Methods

Results

Conclusion