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Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation
H Jaaro1, H Rubinfeld, T Hanoch
1Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, 20024-20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its N-terminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90(RSK), is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.
Insights
Mitogen-activated protein kinase kinase (MEK) translocates to the nucleus upon stimulation. Its nuclear export signal facilitates rapid export, explaining its cytoplasmic presence in resting cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Mitogen-activated protein kinase kinase (MEK) is a cytosolic dual-specificity protein kinase.
- MEK possesses a nuclear export signal (NES) in its N-terminal domain, implying nuclear import and export functions.
- Understanding MEK's localization is crucial for deciphering its role in cellular signaling pathways.
Purpose of the Study:
- To investigate the role of MEK's nuclear export signal in its cellular localization.
- To determine how MEK's localization changes upon mitogenic stimulation.
- To elucidate the mechanisms governing MEK's nuclear-cytoplasmic shuttling.
Main Methods:
- Construction and expression of MEK mutants lacking the nuclear export signal in COS7 and HEK-293T cells.
- Confocal microscopy to visualize the cellular localization of wild-type and mutant MEK.
- In vitro and in vivo assays to assess the substrate specificity of recombinant MEK.
Main Results:
- Deletion of the NES from constitutively active MEK resulted in its constitutive nuclear localization.
- Deletion of the NES from catalytically inactive MEK led to cytoplasmic localization in resting cells, with nuclear translocation upon stimulation.
- Active MEK mutants localized to the nuclear envelope and cell periphery; substrate specificity remained unchanged.
Conclusions:
- MEK undergoes massive nuclear translocation upon mitogenic stimulation.
- The NES is critical for the rapid export of MEK from the nucleus.
- Nuclear export mediated by the NES explains the predominant cytoplasmic localization of wild-type MEK in resting cells.