title: Recruitment of APPL1 to ubiquitin-rich aggresomes in response to proteasomal impairment.
abstract: Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1.
authors.affiliation: International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Pilecka
authors.forename: Lukasz
authors.initials: L
authors.lastname: Sadowski
authors.forename: Yannis
authors.initials: Y
authors.lastname: Kalaidzidis
authors.forename: Marta
authors.initials: M
authors.lastname: Miaczynska
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D007976:Leupeptins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde