title: c-Cbl and Cbl-b ligases mediate 17-allylaminodemethoxygeldanamycin-induced degradation of autophosphorylated Flt3 kinase with internal tandem duplication through the ubiquitin proteasome pathway.
abstract: The class III receptor-tyrosine kinase Flt3 regulates normal hematopoiesis. An internal tandem duplication (ITD) in the juxtamembrane domain of Flt3 (Flt3-ITD) contributes to transformation and is associated with poor prognosis in acute myeloid leukemia. Here, we demonstrate that, as compared with wild-type Flt3 (Flt3-WT), Flt3-ITD more rapidly undergoes degradation through the proteasomal and lysosomal pathways in model hematopoietic 32D cells and in human leukemic MV4-11 cells. The Hsp90 inhibitor 17-allylaminodemethoxygeldanamycin (17-AAG) preferentially induced the polyubiquitination and proteasomal degradation of Flt3-ITD autophosphorylated on Tyr-591 in these cells. The E3 ubiquitin ligases c-Cbl and to a lesser extent Cbl-b facilitated at least partly Lys-48-linked polyubiquitination of autophosphorylated Flt3-ITD when coexpressed in 293T cells. Moreover, c-Cbl and Cbl-b facilitated degradation of Flt3-ITD in 293T cells and significantly enhanced the 17-AAG-induced decline in autophosphorylated Flt3-ITD. The enhancement of Flt3-ITD degradation was also observed in 32D cells inducibly overexpressing c-Cbl or Cbl-b. Furthermore, overexpression of loss-of-function mutants of both c-Cbl (c-Cbl-R420Q) and Cbl-b (Cbl-b-C373A) together in 32D cells retarded the degradation of autophosphorylated Flt3-ITD and significantly inhibited the 17-AAG-induced degradation of Flt3-ITD to confer the resistance to cytotoxicity of 17-AAG on these cells. These results suggest that c-Cbl as well as Cbl-b may play important roles in Hsp90 inhibitor-induced degradation of Flt3-ITD through the ubiquitin proteasome system and in regulation of the basal expression level of Flt3-ITD in leukemic cells.
authors.affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan.
authors.forename: Gaku
authors.initials: G
authors.lastname: Oshikawa
authors.affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan.
authors.forename: Toshikage
authors.initials: T
authors.lastname: Nagao
authors.affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan.
authors.forename: Nan
authors.initials: N
authors.lastname: Wu
authors.affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kurosu
authors.affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan. Electronic address: miura.hema@tmd.ac.jp.
authors.forename: Osamu
authors.initials: O
authors.lastname: Miura
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D016227:Benzoquinones / Q000494:pharmacology*; D045744:Cell Line, Tumor; D015973:Gene Expression Regulation, Leukemic; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D007700:Kinetics; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000502:physiology*; D025801:Ubiquitin / Q000737:chemistry; D051941:fms-Like Tyrosine Kinase 3 / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D016227:Benzoquinones; C118387:Cblb protein, mouse; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D025801:Ubiquitin; C112765:tanespimycin; C492211:CBLB protein, human; D050721:Proto-Oncogene Proteins c-cbl; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D051941:fms-Like Tyrosine Kinase 3; D046988:Proteasome Endopeptidase Complex