The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2.
The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-alpha- and anti-beta-adaptin antibodies but not with anti-gamma-adaptin antibody. In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti-alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein. The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.
Benmerah
A
A
Développement Normal et Pathologique du Système Immunitaire, INSERM U 429; Hôpital Necker-Enfants Malades, Paris, France.
Gagnon
J
J
Bègue
B
B
Mégarbané
B
B
Dautry-Varsat
A
A
Cerf-Bensussan
N
N
D016475:3T3 Cells / Q000201:enzymology; D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033983:Adaptor Protein Complex mu Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D002966:Clathrin; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000166:cytology / Q000201:enzymology*
C499064:AP1M2 protein, human; C510070:AP3M2 protein, human; D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033983:Adaptor Protein Complex mu Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000911:Antibodies, Monoclonal; C438884:Ap1m2 protein, mouse; C510071:Ap3m2 protein, mouse; D002135:Calcium-Binding Proteins; D002966:Clathrin; C486206:EPS15 protein, human; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C499065:adaptor protein complex 1, mu 2 subunit; C438886:adaptor protein complex 2, mu 2 subunit; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases