Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells.

Eisuke Nishida,Makoto Fukuda,Yutaka Matsubayashi

doi:10.1074/jbc.m106012200

Eisuke Nishida, Makoto Fukuda + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m106012200

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Journal: The Journal of biological chemistry	Publication Date: Sep 6, 2001
Citations: 142	License type: cc-by

Affiliation: Kyoto University

Abstract

The classical mitogen-activated protein kinase (MAPK, also known as ERK) pathway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cytoplasm to the nucleus. At least two pathways for the nuclear import of MAPK are shown to exist; passive diffusion of a monomer and Ran-dependent active transport of a dimer, the detailed molecular mechanism of which is unknown. In this study, we have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with GFP-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion. GFP-MAPK was able to accumulate in the nucleus irrespective of its phosphorylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutinin or an excess amount of importin-beta or at low temperatures. Moreover, MAPK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitro. Taken together, these results suggest the third pathway for nuclear import of MAPK, in which MAPK passes through the nuclear pore by directly interacting with the nuclear pore complex.

Highlights

The classical mitogen-activated protein kinase (MAPK,1 known as ERK) cascade is among the key signaling pathways regulating many cellular events, such as cell proliferation, cell differentiation, and early embryonic development
We have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with green fluorescent protein (GFP)-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion
Our experiments identified a novel pathway for nuclear translocation of MAPK, i.e. MAPK can be imported into the nucleus through direct interaction with the nuclear pore complex (NPC) (Fig. 4C)

Summary

EXPERIMENTAL PROCEDURES

Cell Cultures and Transfection—HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum and antibiotics (100 units/ml penicillin and 0.2 mg/ml kanamycin). ⌬BRaf-ER cells [38], a gift from Dr M. Cell Cultures and Transfection—HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum and antibiotics (100 units/ml penicillin and 0.2 mg/ml kanamycin). McMahon, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics. Transfection into HeLa cells and ⌬B-Raf-ER cells was performed using LipofectAMINE and LipofectAMINE PLUS reagents (Life Technologies, Inc.) according to the manufacturer’s instructions with the use of 1 ␮g of total DNA/35 mm dish. DNA Construction—The mammalian expression plasmid harboring Xenopus MAPKK (i.e. MEK) [18] and the bacterial expression plasmids harboring His-tagged Xenopus MAPK [17], His-tagged kinase-dead Xenopus MAPKK [39], His-tagged constitutively active Xenopus MAPKK (LA-SDSE, see Ref. 40), and GST-RanQ69L [19] were constructed previously. To obtain a mammalian expression plasmid harboring GFPMAPK, the open reading frame of Xenopus MAPK (i e. ERK2) was

Analyses of Nuclear Translocation of MAPK in Vitro

RESULTS

DISCUSSION