Life Science April 2001

April 2001 Cell Biology

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 A TECHNICAL APPLICATION NEWSLETTER VOL 2   ISSUE 2    April 2001

 Cell Biology


Leptomycin B: An Important Tool for the Study of Nuclear Export

by Yael Asscher,* Shlomo Pleban,* Marcia Ben-Shushan,* Michal Levin-Khalifa,* Zhong Yao, and Rony Seger
Sigma-Aldrich Corporation,* Jerusalem, Israel and The Weizmann Institute of Science, Rehovot, Israel

Introduction

Export of mRNA and certain proteins from the nucleus is a key step in protein production, proliferation, and apoptosis. An important new tool for the study of nuclear export is the potent antibiotic, leptomycin B, which inhibits the active export of most, if not all, macromolecules from the nucleus. Leptomycin B was first identified in Streptomyces sp. as an unsaturated, branched-chain fatty acid with antifungal activity.1,2 Later, this drug was shown to have antitumor activity,3 to inhibit the G1 and G2 phases of the cell cycle,4 and other cellular functions. These pleotropic effects led to the study of the mechanisms by which leptomycin B influences intracellular processes. One of these studies involved a screen of leptomycin B resistance genes in fission yeast.5 In this screen it was found that leptomycin B targets CRM1 (exportin 1), a fission yeast nuclear protein, which was thought to be involved in the control of gene expr ession. A few years later it was shown that the main function of CRM1 is to export proteins from the nucleus6 by binding to a specific leucine-rich nuclear export signal (NES) of exported proteins and introduce them to the export machinery of the nuclear pores.7 Leptomycin B forms a covalent complex via its , ß-unsaturated -lactone (Figure 1) with the sulfhydryl group of a conserved cysteine residue in CRM1, inhibiting CRM1 interaction with the nuclear localization signal (NLS) of target exported proteins.8,9 This mode of action has been confirmed by mutational analysis.10,1

Today it is known that CRM1 is the main mediator of nuclear export in many cell types. The ability of leptomycin B to inhibit nuclear export has made it a useful tool in the study of the subcellular localization of many regulatory proteins. One of the first proteins whose nuclear export was shown to be blocked by leptomycin B was Rev, which participates in the export of mRNA from the nucleus.12 Other proteins that have been shown to be influenced by leptomycin B, include actin,13 c-Abl,14 Cyclin B1,15 MDM2/p53,16 IkB,17 MPF,18 and PKA.19 Here we describe the purification of leptomycin B from Streptomyces sp. and its use to study the subcellular localization of another important regulatory protein, the mitogen-activated protein kinase (MAPK) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK1), which acts within the MAPK cascade to regulate a large variety of cellular processes includingproliferation, differentiation, development, learning and memory, and hormone action.20

Results and Discussion

Isolation of leptomycin B

Leptomycin B (Figure 1) is an unsaturated, branched-chain fatty acid that is produced in Streptomyces sp.1 together with other similar polyketides such as leptomycin A, kazusamycin A, kazusamycin B, and other alkaloids.21 We applied large-scale reverse-phase chromatography to purify leptomycin B and obtained >95% pure product, as determined by analytical HPLC (Figure 2). The product identity was verified spectroscopically. Thus, mass spectroscopy clearly distinguished the different products obtained in the fermentation and proved that we isolated pure leptomycin B. Negative-ionization Fast Atom Bombardment Mass Spectrometry (FABMS, Finnigan TSQ70) of lept omycin B afforded a molecular ion at m/z 540 [M-H]- and positive-ionization FABMS afforded m/z 564 ion, which corresponds to [M+Na]+. The 300 MHz HNMR spectra (Bruker MX300) of the compound supported these results.

The stability of the product was evaluated in DMSO, in ethanol, or in methanol:water (70:30). It was found that the leptomycin B was unstable in DMSO and decomposed within a few hours. On the other hand, the product was found to be stable in methanol:water solution at room temperature for a few days. For longer storage time, a low temperature such as -20 °C is necessary.

The subcellular distribution of MEK1

One important role of nuclear export signals seems to be the maintenance of cytosolic localization of some regulatory molecules. One example for such a molecule comes from the study of the MAPK kinase, MEK1. As a key component in the MAPK/ERK cascade, MEK1 is one of the main regulators of proliferation, differentiation, and other cellular processes. Change in localization of protein kinases upon stimulation can be an important step in the regulation of the transmitted signals. In resting cells, the components of the MAPK cascade localize to the cell cytosol. Upon extracellular stimulation, there is rapid and dramatic redistribution of components of the cascade to either the plasma membrane or the nucleus.22 MEK1 seems to be one of the molecules that translocates into the nucleus.23 However, after translocation, MEK1 seems to be rapidly exported from the nucleus by its NES23,24 giving rise to its apparent cytosolic localization. Although the timing and the exact role of MEK1 nuclear translocation are still controversial,25,26 one possible function for this translocation could be to facilitate ERK export from the nucleus to the cytoplasm.27 This could explain the importance of the rapid shuttling of MEK1 in and out of the nucleus, which was recently shown to play a role in the regulation of proliferation, differentiation, and even oncogenic transformation of fibroblasts.26,28

Study of the subcellular localization of MEK1 using leptomycin B

In order to better understand the localization of MEK upon mitogenic stimulation, we fused a green fluorescent protein (GFP) to the C-terminus of MEK1. Since the fluorescence of GFP can be easily detected in living cells, this tag serves as a useful tool in the study of cellular localization of many proteins. We transfected the GFP-MEK1 into Rat1 cells, and followed the distribution of the GFP-tagged protein using fluorescence microscopy. Six hours after transfection, the cells were serum-starved, and the localization of GFP-MEK1 was determined at different times. After 14 hours of serum starvation, GFP-MEK1 was homogeneously distributed in the cytosol of transfected cells (Figure 3A, no LMB), and this distribution remained the same for 48-72 hours after serum starvation (data not shown). However, when leptomycin B was added to the cells, the localization of the tagged protein was rapidly changed, and 60 minutes after the addition of 1 to 10 ng/ml leptomycin B to the cells, most of the GFP-MEK1 was detected in the nucleus (Figure 3A). These results are similar to those observed by Nishida and coworkers,27,29 who found that leptomycin B causes nuclear accumulation of MEK in adherent cells.

To examine the potency and specificity of the translocation, we then performed dose-response and time-course studies. Since the translocation processes are not synchronized, the amount of translocated MEK1 could significantly vary between cells at any given time. In order to account for this variation, we counted 100-200 cells for each condition used, and classified the observed MEK1 localization into 4 categories: (i) only cytosolic (C>>N), (ii) mostly cytosolic (C>N), (iii) equally distributed (C=N), and mostly nuclear (C<N) (Figure 3B). Our results indicate that nuclear translocation of MEK1 could be detected when the concentration of leptomycin B used was as low as 0.1 ng/ml at 60 minutes after treatment. The nuclear localization of MEK1 increased in time- and dose- dependent manners. Maximal translocation was observed 120 minutes after treatment with 1 ng/ml leptomycin B (50-80% nuclear). This extent of translocation was not changed even when higher concentrations of leptomycin B were added to the cells, indicating that, as previously observed for ERKs30, a small portion of MEK1 is retained in the cytosol and does not translocate into the nucleus.

Conclusions

These results clearly demonstrate that MEK1 is translocated into the nucleus in the Rat1 cells, and is rapidly exported out of this location by a process inhibited by leptomycin B, indicating that this is an NES- and CRM1-dependent process. These results are similar to those observed with an MEK1 that was mutated to inactivate its NES. Since leptomycin B is a potent inhibitor of the CRM1-dependent nuclear export, MEK translocation can be efficiently studied even with low concentration of leptomycin B without the use of NES-mutants of MEK, which are known to have additional effects on MEK activity. It should be noted, that beside its effect on CRM1, leptomycin B might effect additional intracellular signaling pathways (data not shown). Therefore, more studies are required to elucidate the exact mechanism by which MEK1 enters the nucleus and whether this translocation is activation dependent.

References

1. Hamamoto, T. et al. Leptomycins A and B, new antifungal antibiotics. I. Taxonomy of the producing strain and their fermentation, purification and characterization. J. Antibiot. 36, 639-645 (1983).

2. Hamamoto, T. et al. Leptomycins A and B, new antifungal antibiotics. II. Structure elucidation. J. Antibiot. 36, 646-650 (1983).

3. Komiyama, K. et al. Antitumor activity of leptomycin B. J. Antibiot. 38, 427-429 (1985).

4. Yoshida, M. et al. Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells. Exp. Cell Res. 187, 150-156 (1990).

5. Nishi, K. et al. Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J. Biol Chem. 269, 6320-6324 (1994).

6. Fornerod, M. et al. The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 16, 807-816 (1997).

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8. Kudo, N. et al. Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins. J. Biol. Chem. 272, 29742-29751 (1997).

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10. Neville, M. and Rosbash, M. The NES-Crm1p export pathway is not a major mRNA export route in Saccharomyces cerevisiae. Embo J. 18, 3746-3756 (1999).

11. Kudo, N. et al. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96, 9112-9117 (1999).

12. Wolff, B. et al. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4, 139-147 (1997).

13. Wada, A., et al. Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein. EMBO J. 17, 1635-1641 (1998).

14. Taagepera, S. et al. Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase. Proc. Natl. Acad. Sci. USA 95, 7457-7462 (1998).

15. Yang, J. et al. Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1. Genes Dev. 12, 2131-2143 (1998).

16. Freedman, D. A. and Levine, A. J. Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Mol. Cell Biol. 18, 7288-7293 (1998).

17. Sachdev, S. and Hannink, M. Loss of IkappaB alpha-mediated control over nuclear import and DNA binding enables oncogenic activation of c-Rel. Mol. Cell Biol. 18, 5445-5456 (1998).

18. Hagting, A., et al., MPF localization is controlled by nuclear export. EMBO J. 17, 4127-4138 (1998).

19. Wen, W. et al. Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal. J. Biol. Chem. 269, 32214-32220 (1994).

20. Seger, R. and Krebs, E. G. The MAPK signaling cascade. FASEB J. 9, 726-735 (1995).

21. Stadler, M., et al., Identification of alkaloids and polyketides in an Actinomycete by high-performance liquid chromatography with mass spectrometric and UV-Visible detection. J. Chromatogr. A 818 (2), 187-195 (1998).

22. Rubinfeld, H. et al. Identification of a cytoplasmic-retention sequence in ERK2. J. Biol. Chem. 274, 30349-30352 (1999).

23. Jaaro, H., et al. Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. Proc. Natl. Acad. Sci. USA 94, 3742-3747 (1997).

24. Fukuda, M., et al. Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal. J. Biol. Chem. 271, 20024-20028 (1996).

25. Fukuda, M., et al. A novel regulatory mechanism in the mitogen-activated protein (MAP) kinase cascade. Role of nuclear export signal of MAP kinase kinase. J. Biol. Chem. 272, 32642-32648 (1997).

26. Fukuda, M., et al. Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J. 16, 1901-1908 (1997).

27. Adachi, M., et al. Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J. Cell Biol. 148, 849-856 (2000); published erratum appears in J. Cell Biol. 149(3), 754 (2000).

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About the Authors

Yael Asscher, Ph.D., is manager of R&D, Shlomo Pleban, Ph.D., is the head of Biotechnology R&D department. Marcia Ben-Shushan, Ph.D., is a scientist and Michal Levin-Khalifa, M.Sc., is a food and biotechnology engineer in Biotechnology R&D department at Sigma-Aldrich, Jerusalem, Israel. Rony Seger, Ph.D., is a professor and Zhong Yao is a doctoral student in the Department of Biological Regulation at The Weizmann Institute of Science, Rehovot, Israel.

ORDERING INFORMATION

Product Code

Product Name

Unit

L2913

Leptomycin B95% by HPLC 0.5 µg in 100µl 70% methanol

0.5 µg

   
 
   
 

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