Geldanamycin stimulates internalization of ErbB2 in a proteasome-dependent way.
The potent oncoprotein and receptor tyrosine kinase ErbB2 is remarkable because it resists efficient downregulation. However, ErbB2 can be downregulated by the HSP-90 inhibitor geldanamycin, but the underlying cellular mechanisms are uncertain. Apparently, delivery of ErbB2 to lysosomes, cleavage of the ErbB2 kinase domain and proteasomal activity are all processes that are involved. Using a non-invasive confocal microscopical assay allowing quantitative analysis of ErbB2 internalization in cell populations, we show that whereas ErbB2 is resistant to internalization in untreated SK-BR-3 cells, geldanamycin stimulates internalization and subsequent degradation in lysosomes. This process depends on proteasomal activity, which is a regulatory upstream event in ErbB2 internalization rather than the actual mechanism of degradation. ErbB2 can be internalized as a full-length protein, thus cleavage of the ErbB2 kinase domain is not a requirement for geldanamycin-stimulated internalization. Moreover, as shown by FRAP (fluorescence recovery after photobleaching) and electron microscopy, geldanamycin induces an increase in the amount of mobile ErbB2 and a redistribution of ErbB2 in the plasma membrane making the receptor accessible to endocytosis. Cells with most ErbB2 endocytosis also have the highest fraction of mobile ErbB2. It is concluded that geldanamycin stimulates internalization of full-length ErbB2 in a proteasome-dependent manner leading to lysosomal degradation.
Lerdrup
Mads
M
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, Blegdamsvej 3C, University of Copenhagen, 2200 Copenhagen, Denmark.
Hommelgaard
Anette M
AM
Grandal
Michael
M
van Deurs
Bo
B
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000111:Acetylcysteine; D016227:Benzoquinones; D015853:Cysteine Proteinase Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D046988:Proteasome Endopeptidase Complex; D018719:Erb-b2 Receptor Tyrosine Kinases; C067713:lactacystin; C001277:geldanamycin