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© The Rockefeller University Press, 0021-9525/2000//905 $5.00
The Journal of Cell Biology, Volume 150, Number 4, , 2000 905-912

Tyrosine Phosphorylation of Eps15 Is Required for Ligand-Regulated, but Not Constitutive, Endocytosis

^a Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy
^b Department of Experimental Medicine, Anatomy Section, University of Genova, 16132 Genova, Italy
^c The FIRC Institute for Molecular Oncology (IFOM), 20139 Milan, Italy
Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milan, Italy.39-02-5748985139-02-57489855

pdifiore{at}ieo.it

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

Key Words: Eps15 • phosphotyrosine • endocytosis • epidermal growth factor receptor • transferrin receptor

© 2000 The Rockefeller University Press

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