RESUMEN
The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Transformación Celular Neoplásica/patología , Regulación de la Expresión Génica , Leucemia Experimental/etiología , Proteínas de Ensamble de Clatrina Monoméricas/fisiología , Señales de Exportación Nuclear/genética , Proteínas de Fusión Oncogénica/metabolismo , Secuencia de Aminoácidos , Animales , Antibióticos Antineoplásicos/farmacología , Trasplante de Médula Ósea , Células Cultivadas , Ácidos Grasos Insaturados/farmacología , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Tasa de SupervivenciaRESUMEN
The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.
Asunto(s)
Homeostasis , Hierro/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Aminoácidos/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Clatrina/metabolismo , Embrión de Mamíferos/citología , Endocitosis/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Homeostasis/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Quelantes del Hierro/farmacología , Deficiencias de Hierro , Ratones , Proteínas de Ensamble de Clatrina Monoméricas/química , Proteínas de Ensamble de Clatrina Monoméricas/deficiencia , Fenotipo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismoRESUMEN
beta-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an ankyrin-based mechanism for sarcolemmal localization of dystrophin and beta-DG. Dystrophin binds ankyrin-B and ankyrin-G, while beta-DG binds ankyrin-G. Dystrophin and beta-DG require ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and beta-DG remain intracellular in ankyrin-B-depleted muscle, where beta-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires ankyrin-B for localization of dystrophin/utrophin and beta-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4.
