Distinct recruitment of Eps15 via Its coiled-coil domain is required for efficient down-regulation of the met receptor tyrosine kinase.
Down-regulation of receptor tyrosine kinases (RTK) through receptor internalization and degradation is critical for appropriate biological responses. The hepatocyte growth factor RTK (also known as Met) regulates epithelial remodeling, dispersal, and invasion and is deregulated in human cancers. Impaired down-regulation of the Met RTK leads to sustained signaling, cell transformation, and tumorigenesis, hence understanding mechanisms that regulate this process is crucial. Here we report that, following Met activation, the endocytic adaptor protein, Eps15, is recruited to the plasma membrane and becomes both tyrosine-phosphorylated and ubiquitinated. Recruitment of Eps15 requires Met receptor kinase activity and involves two distinct Eps15 domains. Unlike previous reports for the EGF RTK, which requires the Eps15 ubiquitin interacting motif, recruitment of Eps15 to Met involves the coiled-coil domain of Eps15 and the signaling adaptor molecule, Grb2, which binds through a proline-rich motif in the third domain of Eps15. Expression of the coiled-coil domain is sufficient to displace the wild-type Eps15 protein complex from Met, resulting in loss of tyrosine phosphorylation of Eps15. Knockdown of Eps15 results in delayed Met degradation, which can be rescued by expression of Eps15 WT but not an Eps15 mutant lacking the coiled-coil domain, identifying a role for this domain in Eps15-mediated Met down-modulation. This study demonstrates a new mechanism of recruitment for Eps15 downstream of the Met receptor, involving the coiled-coil domain of Eps15 as well as interaction of Eps15 with Grb2. This highlights distinct regulation of Eps15 recruitment and the diversity and adaptability of endocytic molecules in promoting RTK trafficking.
Parachoniak
Christine A
CA
Departments of Biochemistry, Medicine, and Oncology, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Park
Morag
M
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D054875:Ubiquitination / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met