Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.

TY - JOUR

T1 - Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.

AU - Confalonieri, S

AU - Salcini, A E

AU - Puri, C

AU - Tacchetti, C

AU - Di Fiore, P P

N1 - Keywords: Amino Acid Substitution; Animals; COS Cells; Calcium-Binding Proteins; Cell Line; Cell Membrane; Cell Nucleus; Endocytosis; Ligands; Mice; Mutagenesis, Site-Directed; Phosphoproteins; Phosphorylation; Receptor, Epidermal Growth Factor; Receptors, Transferrin; Recombinant Proteins; Signal Transduction; Transfection; Tyrosine

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

M3 - Journal article

C2 - 10953014

SN - 0021-9525

VL - 150

SP - 905

EP - 912

JO - Journal of Cell Biology

JF - Journal of Cell Biology

IS - 4

ER -