Importin-β1 and Nucleus Transporter Chromosome Region Maintenance 1 (CRM1) Constitute the Nucleocytoplasmic Transport System of STAT3 in Blood-Derived CLL Cells.
Authors: Hazan-Halevy Inbal , Harris David , Liu Zhiming , Ferrajoli Alessandra , Keating Michael J , Estrov Zeev 
Published: April 2020

Abstract
B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western hemisphere. Although several chromosomal and molecular abnormalities have been identified in CLL cells in recent years, the pathogenesis of CLL is still poorly understood. Signal transducer and activator of transcription 3 (STAT3) plays a major role in cellular physiology. Upon exposure to cytokines or growth factors, STAT3 is tyrosine-phosphorylated, migrates to the nucleus, and binds to DNA. Constitutive phosphorylation of STAT3 on tyrosine 705 residues has been found in several solid tumors and hematologic malignancies. Remarkably, CLL is the only disease in which STAT3 is constitutively phosphorylated (p) on serine rather than tyrosine residues (Frank et al. JCI100:3149, 1997). We have recently discovered that serine pSTAT3 translocates to the nucleus, binds to DNA, activates transcription, and plays a major role in the pathogenensis of CLL. Little is known about the transport mechanisms utilized by STAT molecules in fresh leukemia cells, and no data are available on the transport mechanism of serine pSTAT3. Therefore, we sought to identify the nucleocytoplasmic transport system of serine pSTAT3 in CLL cells. In other cellular systems, importin-α3 or -α6 binds to the nuclear localization signal in STAT3, the N terminus of importin-α is directly recognized by importin-β1, and the complex consisting of STAT3, importin-α3 and improtin-β1 transits through the nuclear pore complexes (NPC). To identify which nucleocytoplasmic transport mechanism of serine pSTAT3 is operative in CLL cells, we performed a series of immunoprecipitation experiments with antibodies to STAT3 and importin-β1. We found that STAT3 co-immunoprecipitated with importin-β1 in whole cell, cytoplasmic, and nuclear extracts. We could not determine which member of the importin-α family binds serine pSTAT3 to form a complex with importin-β1 because none of the investigated α-importins (importin-α1, -α3, -α5, -α6, and -α7) co-immunoprecipitated with STAT3. Similar results were obtained when importin-β1 was immunoprecipitated. Unlike the studied α-importins, serine pSTAT3 and STAT3 co-immunoprecipitated with importin-β1. Thus, either an α-importin binds serine pSTAT3 but failed to co-immunoprecipitate, or an unidentified transporter binds serine pSTAT3. After establishing that importin-β1 translocates serine pSTAT3 to the nucleus, we sought to identify the nuclear export mechanism. The established nuclear export mechanism of STAT3 consists of CRM1 that binds to the nuclear export signal on STAT3 and exports STAT3 through the NPC. Using an identical experimental design, we immunoprecipitated whole cell, cytoplasmic, and nuclear extracts with anti-CRM1 antibodies and found that STAT3 and serine pSTAT3 co-immunoprecipitated with CRM1. Then, we immunoprecipitated the cell extracts with anti-STAT3 antibodies. In these experiments, CRM1 co-immunoprecipitated with STAT3. To further elucidate the role of CRM1 in the STAT3 nuclear export system, we incubated CLL cells with increasing concentrations of the CRM1 inhibitor leptomycin B and assessed STAT3 protein levels in nuclear extracts by Western immunoblotting. We found that leptomycin B increased the accumulation of STAT3 in the nucleus in a dose dependent manner, further confirming that CRM1 exports STAT3 from the nucleus to the cytoplasm. Taken together, our data demonstrate for the first time that in CLL cells STAT3 and serine pSTAT3 are transported into the nucleus by importin-β1 and exported by CRM1. Targeting this nuclear trafficking system might provide a new therapeutic strategy for the treatment of CLL.