title: Clathrin assembly protein CALM plays a critical role in KIT signaling by regulating its cellular transport from early to late endosomes in hematopoietic cells.
abstract: CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage-Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM-/- mice. Also, colony forming activity was impaired in CALM-/- LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM-/- LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM-/- murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM-/- MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM-/- MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM-/- MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM-/- MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM-/- KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.
authors.affiliation: Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Rai
authors.affiliation: Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan; Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women's University, Nara, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women's University, Nara, Japan.
authors.forename: Honami
authors.initials: H
authors.lastname: Ogoh
authors.affiliation: Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Taniguchi
authors.affiliation: Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Morita
authors.affiliation: Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shimada
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Tanimura
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Matsui
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Yokota
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oritani
authors.affiliation: Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tanabe
authors.affiliation: Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women's University, Nara, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Kanakura
authors.affiliation: Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
authors.forename: Itaru
authors.initials: I
authors.lastname: Matsumura
mesh_terms: D000818:Animals; D017209:Apoptosis; D001692:Biological Transport; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D034041:Monomeric Clathrin Assembly Proteins / Q000502:physiology*; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology*; D015398:Signal Transduction
chemical_list: D034041:Monomeric Clathrin Assembly Proteins; C573094:PICALM protein, mouse; D019009:Proto-Oncogene Proteins c-kit