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

The classical mitogen-activated protein kinase (MAPK, also known as ERK) pa thway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cy toplasm to the nucleus. At least two pathways for the nuclear import of MAP K are shown to exist; passive diffusion of a monomer and Ran-dependent acti ve transport of a dimer, the detailed molecular mechanism of which is unkno wn. 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 phospho rylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutini n or an excess amount of importin-beta or at low temperatures. Moreover, AL APK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitr o. Taken together, these results suggest the third pathway for nuclear impo rt of MAPK, in which MAPK passes through the nuclear pore by directly inter acting with the nuclear pore complex.