Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells

<div><p>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 <i>CALM</i>-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 <i>CALM</i>^−/− mice. Also, colony forming activity was impaired in <i>CALM^−/−</i> LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in <i>CALM^−/−</i> LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using <i>CALM</i>^−/− murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in <i>CALM</i>^−/− MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in <i>CALM</i>^−/− MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in <i>CALM</i>^−/− MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in <i>CALM</i>^−/− MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in <i>CALM</i>^−/− 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.</p>