Specific Grb2-mediated Interactions Regulate Clathrin-dependent Endocytosis of the cMet-tyrosine Kinase

Abstract

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.

Endocytosis of Receptor Tyrosine Kinases

“…Goh and A. Sorkin mammalian isoforms of Cbl (Cbl-b, Cbl-3, and c-Cbl) bind directly to specific phosphorylated tyrosines in RTKs, and long Cbl species (c-Cbl and Cbl-b) bind EGFR, c-Met, PDGFR-b, stem cell factor receptor (c-kit), and other RTKs indirectly through the SH2 adaptor protein Grb2 (Batzer et al 1994;Peschard et al 2001;Waterman et al 2002;Li et al 2007;Sun et al 2007). The key role of Grb2 in ubiquitination and internalization of RTKs via Cbl has been shown using multiple experimental approaches in mammalian cells (Peschard 2001;Huang and Sorkin 2005;Johannessen 2006;Li 2007). In Drosophila and Caenorhabditis elegans, D-Cbl L binds to Grb2/Drk and Sli-1/Cbl binds to Grb2/SEM-5 to promote EGFR endocytosis (Yoon et al 2000;Wang and Pai 2011).…”

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

“…In addition to its role in cell proliferation, EGFR activation can promote cell survival and differentiation and also drive the profound remodelling of the cell cytoskeleton, inducing migratory and invasive phenotypes in many cell types. The adaptor protein Grb2 is likely to represent a key node coupling EGFR activation to this diverse range of biological outputs as it provides a molecular bridge between active receptor and an array of downstream signalling protein classes including exchange factors, phosphatases, ubiquitin ligases and cytoskeletal remodelling factors [48][49][50][51]. Grb2 is recruited to activated receptor via its Src Homology 2 (SH2) domain which targets specific peptide consensuses flanking a critical central phosphotyrosine residue within the receptor's intracellular domain.…”

GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding

“…GRB2 is a signaling adaptor protein ubiquitously expressed in all embryonic and adult tissues (14) and has been shown to couple epidermal growth factor (EGF) receptor (EGFR) and hepatocyte growth factor receptor activation to downstream signaling and receptor internalization (25,27). Here we show that GRB2 is also important for eMLV entry and infection.…”

“…To gain further insight into the molecular role of GRB2 in eMLV entry, we tested whether mCAT-1 and GRB2 interact. Glutathione S-transferase (GST)-GRB2 fusion proteins (wild-type GRB2 and portions of different functional domains) were used previously to identify and characterize proteins that interact with GRB2 (4,25). A nonionic detergent (n-octyl-␤-D-glucopyranoside)-containing buffer was mod-…”