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. 1997 Apr 15;94(8):3742-7.
doi: 10.1073/pnas.94.8.3742.

Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation

H Jaaro  1 , H Rubinfeld, T Hanoch, R Seger
Affiliations

Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation

H Jaaro et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
Schematic representation of MEK1. The sites of mutations are indicated.
Figure 2
Figure 2
Localization of the overexpressed ΔN-EE-, WT-, and ΔN-KA-EE (ΔN-KA)- MEKs in COS7 cells. COS7 cells transfected with each one of these constructs were subjected to serum starvation for 16 hr and then either stimulated with 10% FCS (10 min), or epidermal growth factor (EGF; 50 μg/ml, 5 min), or left untreated (basal). The cells were stained with anti-MEK1 antibodies and visualized using conventional fluorescent microscopy (Zeiss microscope magnification of ×800).
Figure 3
Figure 3
Localization of overexpressed ΔN-EE-, WT-, ΔN-KA-EE-, EE-, and KA-MEKs in HEK-293T cells. Staining was assessed by conventional fluorescent microscopy as in Fig. 2.
Figure 4
Figure 4
Subcellular localization of ΔN-EE-, WT-, and ΔN-KA-EE-MEKs in COS7 cells by confocal microscopy. The slides used in Fig. 2 were viewed by confocal microscope. (Bio-Rad MRC-1024, objective ×20, zoom 3.6.)
Figure 5
Figure 5
Determination of the cellular localization of ΔN-EE-, WT-, and ΔN-KA-EE-MEKs in COS7 cells using fractionation. COS7 cells overexpressing the constructs were either left untreated (basal control), or stimulated with PMA (250 nM, 10 min). The cells were fractionated and subjected to (A) Western blotting with monoclonal anti-MEK1 antibodies (due to differences in amounts of overexpression, the amount of MEK in this figure can only be compared in the basal and stimulated pairs that were derived from the same transfection), or (B) assay of MAPKK activity as previously described (16). These results are the average of three distinct experiments. VC, vector control.
Figure 6
Figure 6
Substrate specificity of bacterially expressed 6His-ΔN-EE-MEK. 6His-ΔN-EE and 6His-WT-MEKs were expressed, purified, and subjected to Coomassie-blue staining (A Left). The two constructs (0.2 μg each) were used for in vitro phosphorylation (20 min, 30°C) of 0.1 μg, 0.5 μg, or no 6His-ERK2 (A Center). In a separate experiment, 6His-ΔN-EE-MEK (0.25 μg) was incubated under the same conditions with 6His-JNK1 (1 μg), 6His-p38RK (1 μg), or buffer alone. As control, the 6His-JNK1 and 6His-p38RK were incubated with buffer alone (A Right). W and ΔN indicate 6His-WT- and 6His-ΔN-EE-MEKs, respectively. (B) The proteins protamine, casein, histone (type III Sigma), nuclear K7 protein (gift from K. Bomsztyk, University of Washington), denatured extract (70°C, 10 min) of COS7 cells (de-cytosol, 10 μg), denatured (56°C, 10 min) ERK2 (de-ERK2, 0.5 μg), MBP, catalytically inactive ERK1 (6His-KR-ERK1, 0.5 μg), 6His-ERK2 (0.25 μg), or buffer A were subjected to in vitro phosphorylation by 6His-ΔN-EE-MEK (0.2 μg). The results in A and B were repeated three times.
Figure 7
Figure 7
ΔN-EE-MEK stimulates exclusively the ERK cascade in COS7 cells. Three HA-tagged MAPK isoforms (ERK2, JNK2, and p38RK) were cotransfected to COS7 cells with or without ΔN-EE-MEK followed by PMA stimulation (as in Fig. 5), immunoprecipitation using anti-HA antibodies, and kinase assay. The amount of phosphate incorporated into MBP in the stimulated ERK2 was 95-fold more than that in p38RK and 105-fold of that in c-Jun. This is a representative experiment that was repeated four times.
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