ERK2 enters the nucleus by a carrier-independent mechanism.
In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.
Whitehurst
Angelique W
AW
Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9041, USA.
Wilsbacher
Julie L
JL
You
Youngjai
Y
Luby-Phelps
Kate
K
Moore
Mary Shannon
MS
Cobb
Melanie H
MH
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001081:Apyrase / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004734:Energy Metabolism; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D028884:Karyopherins / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D000097606:Exportin 1 Protein
D001081:Apyrase; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014909:Wheat Germ Agglutinins; D000097606:Exportin 1 Protein