Overexpression of the Nucleoporin CAN/NUP214 Induces Growth Arrest, Nucleocytoplasmic Transport Defects, and Apoptosis

ABSTRACT

The human CAN gene was first identified as a target of t(6;9)(p23;q34), associated with acute myeloid leukemia and myelodysplastic syndrome, which results in the expression of aDEK-CAN fusion gene. CAN, also called NUP214, is a nuclear pore complex (NPC) protein that contains multiple FG-peptide sequence motifs. It interacts at the NPC with at least two other proteins, the nucleoporin NUP88 and hCRM1 (exportin 1), which was recently shown to function as a nuclear export receptor. Depletion of CAN in knockout mouse embryonic cells results in cell cycle arrest in G₂, followed by inhibition of nuclear protein import and a block of mRNA export. We overexpressed CAN and DEK-CAN in U937 myeloid precursor cells. DEK-CAN expression did not interfere with terminal myeloid differentiation of U937 cells, whereas CAN-overexpressing cells arrested in G₀, accumulated mRNA in their nuclei, and died in an apoptotic manner. Interestingly, we found that hCRM1 and import factor p97/importin β colocalized with the ectopically expressed CAN protein, resulting in depletion of both factors from the NPC. Overexpression of the C-terminal FG-repeat region of CAN, which contains the binding site for hCRM1, caused sequestering of hCRM1 in the nucleoplasm and was sufficient to inhibit cell growth and to induce apoptosis. These results confirm that CAN plays a crucial role in nucleocytoplasmic transport and imply an essential role for hCRM1 in cell growth and survival.

ACKNOWLEDGMENTS

We are grateful to Dario Vignali for pUHD/TetVP16Puro, Gabriel Nuñez for the spleen focus-forming virus Bcl-x_L plasmid, John Cleveland for a c-mycprobe, Charles Sherr for a cyclin D₂ probe, Stephen Adam for monoclonal antibody MAb318, and Maarten Fornerod for affinity-purified hCRM1 antibodies. We thank Sharon Frase and Andrea Elberger for use of the Confocal Laser Scanning Facility, UT Memphis (funded by PHS grant CLSM 1S10RR08385), Donna Davis and Gopal Murti for electron microscopic studies, Richard Ashmun for FACS analyses, Sjozef van Baal for help with the figures, Charlette Hill for secretarial assistance, and Sue Vallance for scientific editing.

These studies were supported in part by Cancer Center CORE grant CA-21765 and by the Associated Lebanese Syrian American Charities (ALSAC) of St. Jude Children’s Research Hospital.