Examining the mechanism of Erk nuclear translocation using green fluorescent protein.
In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2.
Horgan
Angela M
AM
Vollum Institute for Advanced Biomedical Research, L474, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA.
Stork
Philip J S
PJ
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019281:Dimerization; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology*; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases