title: Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylamino-demethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70-interacting protein.
abstract: Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a constitutively active fusion tyrosine kinase involved in lymphomagenesis of human anaplastic large cell lymphomas (ALCL), the maturation and activity of which depend on the association with the heat shock protein (hsp) 90 protein chaperone. Targeting hsp90 by the ansamycins geldanamycin and 17-allyl-amino-demethoxygeldanamycin (17-AAG) promotes degradation of several proteins through the ubiquitin-proteasome pathway, including oncogenic Raf, v-Src, erbB2, and BCR-ABL. We have previously shown that 17-AAG prevents hsp90/NPM-ALK complex formation and fosters NPM-ALK turnover, perhaps through its association with the hsp70 chaperone. Here, we show that inhibition of the proteasome activity by the potent and specific compound pyrazylcarbonyl-Phe-Leu-boronate (PS-341) blocks 17-AAG-induced down-regulation of NPM-ALK, which becomes detergent-insoluble and relocates into ubiquitin-rich perinuclear vesicles that represent aggregated polyubiquitinated forms of the protein. Kinase activity was not mandatory for proteasomal degradation of NPM-ALK, because kinase-defective NPM-ALK was even more rapidly degraded upon 17-AAG treatment. Prolonged exposure to the proteasome inhibitor was shown to trigger caspase-3-mediated apoptosis in proliferating ALCL cells at nanomolar concentrations. However, we verified that the accumulation of detergent-insoluble NPM-ALK in ALCL cells was not a spurious consequence of PS341-committed apoptosis, because caspase inhibitors prevented poly(ADP-ribose) polymerase cleavage whereas they did not affect partitioning of aggregated NPM-ALK. In line with these observations, the carboxyl hsp70-interacting ubiquitin ligase (CHIP), was shown to increase basal ubiquitination and turnover of NPM-ALK kinase, supporting a mechanism whereby NPM-ALK proceeds rapidly toward hsp70-assisted ubiquitin-dependent proteasomal degradation, when chaperoning activity of hsp90 is prohibited by 17-AAG.
authors.affiliation: Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, Padova, Italy. paolobonvini@hotmail.com
authors.forename: Paolo
authors.initials: P
authors.lastname: Bonvini
authors.forename: Henry
authors.initials: H
authors.lastname: Dalla Rosa
authors.forename: Nadia
authors.initials: N
authors.lastname: Vignes
authors.forename: Angelo
authors.initials: A
authors.lastname: Rosolen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D016227:Benzoquinones; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D019556:COS Cells; D002458:Cell Fractionation; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D015536:Down-Regulation; D006801:Humans; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*
chemical_list: D016227:Benzoquinones; D001897:Boronic Acids; D003902:Detergents; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011719:Pyrazines; D025801:Ubiquitin; D017828:Rifabutin; C112765:tanespimycin; D000069286:Bortezomib; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex