CIN85 Participates in Cbl-b-mediated Down-regulation of Receptor Tyrosine Kinases

Stanley Lipkowitz,Ana Dinarina,Iwona Szymkiewicz,Philippe Soubeyran,Ivan Dikic,Katarzyna Kowanetz

doi:10.1074/jbc.m205535200

Stanley Lipkowitz, Ana Dinarina + Show 4 more

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https://doi.org/10.1074/jbc.m205535200

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Abstract

The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.

Highlights

The mammalian Cbl protein family consists of three members: Cbl, Cbl-b, and Cbl-3, all having a highly conserved amino-terminal part composed of a tyrosine kinase-binding module
Yeast clones expressing either three SH3 domains of CIN85 (3SH3), a proline-rich region with coiled-coil domain (PCc), or the full size CIN85 were transformed with the carboxyl-terminal part of Cbl-b, and interactions were checked by the ability of clones to grow on selective agars and by filter-lift assays
We have mutated the first proline in these PXXP motifs, each separately or all of them together, and showed that neither mutation abrogated binding of CIN85 to Cbl-b, suggesting that CIN85 binding to Cbl-b may be mediated by PXXP-independent motifs present in the distal carboxyl terminus of Cbl-b

Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 277, No 42, Issue of October 18, pp. 39666 –39672, 2002 Printed in U.S.A. CIN85 Participates in Cbl-b-mediated Down-regulation of Receptor Tyrosine Kinases*. Several recent reports have implicated the carboxyl terminus of Cbl in the control of endocytosis of RTKs. The major mechanism of Cbl recruitment to activated EGF receptors involves binding of the SH2 domain of Cbl to the autophosphorylated tyrosine 1045 of EGF receptor [15]. Binding of SH3 domain-containing protein CIN85 to the distal carboxyl terminus of Cbl was shown to regulate EGF and c-Met receptor endocytosis in mammalian cells [1, 17]. CIN85 and Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in dermatofibrosarcoma protuberans (DFSP), A431 breast cancer, and HMC-1 mastocytoma tumor cell lines, respectively This indicates that Cbl-b and CIN85 participate both in ligand-dependent down-regulation of RTKs and in internalization of constitutively active RTKs found in tumors.

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