Abstract
The nuclear–cytoplasmic distribution of ERK2 is regulated in response to various stimuli and changes in cell context. Furthermore, the nuclear flux of ERK2 occurs by several energy- and carrier-dependent and -independent mechanisms. ERK2 has been shown to translocate into and out of the nucleus by facilitated diffusion through the nuclear pore, interacting directly with proteins within the nuclear pore complex, as well as by karyopherin-mediated transport. Nuclear export has been suggested to be CRM1- and MEK1/2-dependent. Here, we describe a general nuclear import assay of wild-type ERK2 that can be employed to identify different mechanisms governing nuclear entry of the protein kinase, adapted to evaluate ERK2 mutants that impair nuclear entry to dissect energy- and carrier-dependent and -independent mechanisms, and extended to characterize export mechanisms.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Whitehurst, A. W., Cobb, M. H., and White, M. A. (2004). Stimulus-coupled spatial restriction of extracellular signal-regulated kinase1/2 activity contributes to the specificity of signal-response pathways. Mol Cell Biol 24, 10145–10150.
Chen, R. Y., Sarnecki, C., and Blenis, J. (1992). Nuclear localization and regulation of erk and rsk-encoded protein kinases. Mol Cell Biol 12, 915–927.
Matsubayashi, Y., Fukuda, M., and Nishida, E. (2001). Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells. J Biol Chem 276, 41755–41760.
Whitehurst, A. W., Wilsbacher, J. L., You, Y., Luby-Phelps, K., Moore, M. S., and Cobb M. H. (2002). ERK2 enters the nucleus by a carrier-independent mechanism. Proc Natl Acad Sci U S A 99, 7496–7501.
Costa, M., Marchi, M., Cardarelli, F., Roy, A., Beltram, F., Maffei, L., and Ratto, G. M. (2006). Dynamic regulation of ERK2 nuclear translocation and mobility in living cells. J Cell Sci 119, 4952–4963.
Burack, W. R. and Shaw, A. S. (2005). Live cell imaging of ERK and MEK: Simple binding equilibrium can explain ERK’s regulated nucleocytoplasmic distribution. J Biol Chem 280, 3832–3837.
Ranganathan, A., Yazicioglu, M. N., and Cobb, M. H. (2006). The nuclear localization of ERK2 occurs by mechanisms both independent of and dependent on energy. J Biol Chem 281, 15645–15652.
Chuderland, D., Konson, A., and Seger, R. (2008). Identification and characterization of a general nuclear translocation signal in signaling proteins. Mol Cell 31, 850–861.
Phelps, M., Phillips, A., Darley, M., and Blaydes, J. P. (2005). MEK–ERK signaling controls Hdm2 oncoprotein expression by regulating hdm2 mRNA export to the cytoplasm. J Biol Chem 280, 16651–16658.
Yoon, S. O., Shin, S., Liu, Y., Ballif, B. A., Woo, M. S., Gygi, S. P., and Blenis, J. (2008). Ran-binding protein 3 phosphorylation links the Ras and PI3-kinase pathways to nucleocytoplasmic transport. Mol Cell 29, 362–375.
Adachi, M., Fukuda, M., and Nishida E. (2000). Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J Cell Biol 148, 849–856.
Reszka, A. A., Seger, R., Diltz, C. D., Krebs, E. G., and Fischer, E. H. (1995) Association of mitogen-activated protein kinase with the microtubule cytoskeleton. Proc Natl Acad Sci U S A 92, 8881–8885.
Gonzalez, J. M., Navarro-Puche, A., Casar, B., Crespo, P., and Andres, V. (2008) Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope. J Cell Biol 183, 653–666.
Casar, B., Sanz-Moreno, V., Yazicioglu, M. N., Rodriguez, J., Bercioano, M. T., Lafarga, M., Cobb, M. H., and Crespo, P. (2007). Mxi2 promotes stimulus-independent ERK nuclear translocation. EMBO J 26, 635–646.
Vomastek, T., Iwanicki, M. P., Burack, W. R., Tiwari, D., Kumar, D., Parsons, J. T., Weber, M. J., and Nandicoori, V. K. (2008). Extracellular signal-regulated kinase (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2–Tpr interaction. Mol Cell Biol 28, 6954–6966.
Yazicioglu, M. N., Goad, D. L., Ranganathan, R., Whitehurst, A. W., Goldsmith, E. J., and Cobb, M. H. (2007). Mutations in ERK2 binding sites affect nuclear entry. J Biol Chem 282, 28759–28767.
Lee, T., Hoofnagle, A. N., Kabuyama, Y., Stroud, J., Min, X., Goldsmith, E. J., Chen, L., Resing, K. A., and Ahn, N. G. (2004). Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry. Mol Cell 14, 43–55.
Colbeau, A., Nachbaur, J., and Vignais, P.M. (1971). Enzymatic characterization and lipid composition of rat liver subcellular membranes. Biochim Biophys Acta 249, 462–492.
Moore, M. S. and Blobel, G. (1992). The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell 69, 939–950.
Melchior, F., Paschal, B., Evans, J., and Gerace, L. (1993). Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol 123, 1649–1659.
Khokhlatchev, A., Canagarajah, B., Wilsbacher, J. L., Robinson, M., Atkinson, M., Goldsmith, E., and Cobb, M. H. (1998). Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93, 605–615.
Acknowledgments
We would like to acknowledge grant DK34128 from the National Institutes of Health and I1243 from the Welch Foundation for support of work from the Cobb Laboratory.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Meida, LLC
About this protocol
Cite this protocol
Jivan, A., Ranganathan, A., Cobb, M.H. (2010). Reconstitution of the Nuclear Transport of the MAP Kinase ERK2. In: Seger, R. (eds) MAP Kinase Signaling Protocols. Methods in Molecular Biology, vol 661. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-795-2_16
Download citation
DOI: https://doi.org/10.1007/978-1-60761-795-2_16
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-794-5
Online ISBN: 978-1-60761-795-2
eBook Packages: Springer Protocols