Identification of functional nuclear export sequences in human sphingosine kinase 1

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Abstract

Sphingosine kinase (SPHK) is an enzyme that phosphorylates sphingosine to form sphingosine 1-phosphate (S1P). Human SPHK1 (hSPHK1) was localized predominantly in the cytoplasm when transiently expressed in Cos7 cells. In this study, we have found two functional nuclear export signal (NES) sequences in the middle region of hSPHK1. Deletion and mutagenesis studies revealed that the cytoplasmic localization of SPHK1 depends on its nuclear export, directed by the NES. Furthermore, upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of hSPHK1 was observed, indicating that hSPHK1 shuttles between the cytoplasm and the nucleus. Our results provide the first evidence of the active nuclear export of SPHK1 and suggest it is mediated by a CRM1-dependent pathway.

Section snippets

Materials and methods

Materials. Leptomycin B was a kind gift from Dr. M. Yoshida (RIKEN, Discovery Research Institute, Japan). Anti-HA Y-11 polyclonal antibody was purchased from Santa Cruz Biotechnology and the anti-HA HA-7 monoclonal antibody was from Sigma. Alexa 488-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG were from Molecular Probes. DAPI was from Boehringer–Mannheim. Bovine serum albumin (BSA, fraction V) was from Sigma (St. Louis, MO). All other reagents were of the highest purity

A specific region of hSPHK1 is required for its cytoplasmic localization in Cos7 cells

We analyzed the mechanisms controlling the cytoplasmic localization of hSPHK1 in Cos7 cells. To determine which region of the protein is required for the cytoplasmic localization of hSPHK1, N-terminal deletion mutants of hSPHK1 were used (Fig. 1A). Expression vectors encoding N-terminally HA-tagged forms of hSPHK1 (HA-hSPHK1) or the deletion mutants were transfected into Cos7 cells, and their subcellular localization was analyzed by fluorescence microscopy (Fig. 1B). HA-hSPHK1, HA-hSPHK1 ΔN141,

Discussion

In this study, we identified two functional NES sequences in hSPHK1. Deletion and mutagenesis studies revealed that the cytoplasmic localization of SPHK1 depends on its nuclear export, directed by the NES. LMB treatment induced SPHK1 accumulation in the nuclei. Thus, a CRM1-dependent pathway might mediate the nuclear export of SPHK1. Although we could not predict a distinct nuclear localization signal (NLS) in hSPHK1 using database searches (http://ca.expasy.org/prosite/, //cubic.bioc.columbia.edu/predictnls/

Acknowledgements

We thank Dr. M. Yoshida (Chemical Genetics Laboratory, Discovery Research Institute, RIKEN, Japan) for providing leptomycin B, and Dr. A. Kihara and Dr. Y. Mizutani for helpful discussions. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (B) (12140201) from the Ministry of Education, Culture, Sports, Science and Technology.

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    Abbreviations: SPHK, sphingosine kinase; S1P, sphingosine 1-phosphate; NES, nuclear export signal; NLS, nuclear localization signal; CRM1, chromosomal region maintenance 1; LMB, leptomycin B; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; GFP, green fluorescent protein.

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