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A Chimeric Pre‐ubiquitinated EGF Receptor is Constitutively Endocytosed in a Clathrin‐Dependent, but Kinase‐Independent Manner
Author(s) -
Bertelsen Vibeke,
Sak Malgorzata Magdalena,
Breen Kamilla,
Rødland Marianne S.,
Johannessen Lene E.,
Traub Linton M.,
Stang Espen,
Madshus Inger Helene
Publication year - 2011
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2011.01162.x
Subject(s) - clathrin , endocytosis , microbiology and biotechnology , biology , signal transducing adaptor protein , internalization , ubiquitin ligase , fusion protein , ubiquitin , tyrosine kinase , phosphorylation , kinase , signal transduction , biochemistry , receptor , recombinant dna , gene
The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA‐mediated knock‐down of c‐Cbl and Cbl‐b significantly slowed clathrin‐dependent internalization of activated wild‐type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin‐coated pits. However, a chimeric protein consisting of wt‐EGFR, a C‐terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin‐dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over‐rides the requirement for kinase activity in recruitment of the EGFR to clathrin‐coated pits.