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.

Chlorpromazine eliminates acute myeloid leukemia cells by perturbing subcellular localization of FLT3-ITD and KIT-D816V

“…1a). In this condition, in accord with our previous report 24 , FL-induced phosphorylation of FLT3 WT, SCF-induced phosphorylation of KIT WT, and phosphorylation of their downstream molecules (STAT5, ERK, and Akt) were not impaired, while phosphorylation of Akt was slightly augmented and prolonged in Ba/F3-FLT3 WT and Ba/F3-KIT WT by CALM KD ( Supplementary Fig. 1b).…”

“…These findings are largely consistent with the phenotypes of fit1 mice harboring a nonsense point mutation in the PICALM gene 22,23 . Furthermore, we showed that CALM is essential for CCV formation and plays an important role in the intracellular trafficking of KIT from early to late endosomes in hematopoietic cells 24 . In this study, we also found that KIT-mediated cellular growth was partially impaired in CALM −/− HSC/HPCs.…”

Role of Alström syndrome 1 in the regulation of blood pressure and renal function

“…that ALMS1 may be involved in lipid raft-dependent NKCC2 endocytosis. However, we also pulled down phosphatidylinositol binding clathrin assembly protein (PICALM) ( Figure 1E), which mediates clathrin-dependent endocytosis and targeting of clathrin-coated vesicles to early and late endosomes (47). Together, our data suggest that ALMS1 may be a common scaffold that recruits proteins involved in determining lipid raft-and/or clathrin-dependent endocytosis.…”

A novel system-level approach using RNA-sequencing data identifies miR-30-5p and miR-142a-5p as key regulators of apoptosis in myocardial infarction

“…Some recent studies found that Picalm is related to apoptosis and that apoptosis was increased in Picalm -deficient erythroblasts 26 . SCF-induced KIT trafficking was severely impaired in CALM -deficient MEFs leading to the excessive activation of Akt 27 . Knockdown of CALM stimulated autophagosome formation but overexpression decreased autophagy, which protects cells against proapoptotic insults by reducing caspase activation 28 .…”