Abstract
The Ras-controlled extracellular signal-regulated kinase (ERK) pathway mediates a large number of cellular events, from proliferation to survival, from synaptic plasticity to memory formation. In order to study the role of the two major ERK isoforms in the brain, ERK1 and ERK2, we have generated GFP fusion proteins of both protein kinases. In addition, we have produced two swapped constructs in which the N-term tail of ERK1, the domain responsible for its unique properties, has either been removed from ERK1 or attached to ERK2. We demonstrated that all four GFP proteins are properly expressed in vitro in mouse embryo fibroblasts. However, only ERK1 and ERK2 > 1 overexpression resulted in a significant growth retardation. In addition, we have expressed all four GFP fusion constructs in vivo, in the adult brain, using lentiviral vector-assisted transgenesis and found that they are predominantly neuronal. Finally, we have devised an ex-vivo system, in which brain slices prepared from adult mice can be stimulated with glutamate and the activation of both cytoplasmic and nuclear substrates of ERK can be detected. Since phosphorylation of both the ribosomal protein S6 and of the histone H3 is completely prevented by a chemical inhibition of the ERK pathway, this ex-vivo system can be exploited in the future to investigate the regulation of the ERK cascade using slices from LV-injected or conventional transgenic animals.
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
References
Pearson G et al (2001) Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22(2):153–183
Rubinfeld H, Seger R (2005) The ERK cascade: a prototype of MAPK signaling. Mol Biotechnol 31(2):151–174
Shaul YD, Seger R (2006) The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta 1773(8):1213–1226
Ramos JW (2008) The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. Int J Biochem Cell Biol 40(12):2707–2719
Orban PC, Chapman PF, Brambilla R (1999) Is the Ras-MAPK signalling pathway necessary for long-term memory formation? Trends Neurosci 22(1):38–44
Chuderland D, Seger R (2005) Protein-protein interactions in the regulation of the extracellular signal-regulated kinase. Mol Biotechnol 29(1):57–74
Vantaggiato C et al (2006) ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially. J Biol 5(5):14
Thomas GM, Huganir RL (2004) MAPK cascade signalling and synaptic plasticity. Nat Rev 5(3):173–183
Sweatt JD (2004) Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 14(3):311–317
Davis S, Laroche S (2006) Mitogen-activated protein kinase/extracellular regulated kinase signalling and memory stabilization: a review. Genes Brain Behav 5(Suppl 2):61–72
Samuels IS, Saitta SC, Landreth GE (2009) MAP’ing CNS development and cognition: an ERKsome process. Neuron 61(2):160–167
Santini E, Valjent E, Fisone G (2008) Parkinson’s disease: levodopa-induced dyskinesia and signal transduction. FEBS J 275(7):1392–1399
Girault JA, Valjent E, Caboche J, Herve D (2007) ERK2: a logical AND gate critical for drug-induced plasticity? Curr Opin Pharmacol 7(1):77–85
Kim EK, Choi EJ (2010) Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta 1802(4):396–405
Tidyman WE, Rauen KA (2009) The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 19(3):230–236
Klann E, Dever TE (2004) Biochemical mechanisms for translational regulation in synaptic plasticity. Nat Rev 5(12):931–942
Marchi M et al (2008) The N-terminal domain of ERK1 accounts for the functional differences with ERK2. PLoS One 3(12):e3873
Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 92(17):7686–7689
Favata MF et al (1998) Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273(29):18623–18632
Sebolt-Leopold JS, Herrera R (2004) Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer 4(12):937–947
Saba-El-Leil MK et al (2003) An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep 4(10):964–968
Yao Y et al (2003) Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation. Proc Natl Acad Sci U S A 100(22):12759–12764
Hatano N et al (2003) Essential role for ERK2 mitogen-activated protein kinase in placental development. Genes Cells 8(11):847–856
Meloche S, Vella FD, Voisin L, Ang SL, Saba-El-Leil M (2004) Erk2 signaling and early embryo stem cell self-renewal. Cell Cycle 3(3):241–243
Fischer AM, Katayama CD, Pages G, Pouyssegur J, Hedrick SM (2005) The role of erk1 and erk2 in multiple stages of T cell development. Immunity 23(4):431–443
Satoh Y et al (2007) Extracellular signal-regulated kinase 2 (ERK2) knockdown mice show deficits in long-term memory; ERK2 has a specific function in learning and memory. J Neurosci 27(40):10765–10776
Pagès G et al (1999) Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 286(5443):1374–1377
Selcher JC, Nekrasova T, Paylor R, Landreth GE, Sweatt JD (2001) Mice lacking the ERK1 isoform of MAP kinase are unimpaired in emotional learning. Learn Mem 8(1):11–19
Mazzucchelli C et al (2002) Knockout of ERK1 MAP kinase enhances synaptic plasticity in the striatum and facilitates striatal-mediated learning and memory. Neuron 34:807–820
Indrigo M, Papale A, Orellana D, Brambilla R (2010) Lentiviral vectors to study the differential function of ERK1 and ERK2 MAP kinases. Methods Mol Biol 661:205–220
Tronson NC et al (2008) Regulatory mechanisms of fear extinction and depression-like behavior. Neuropsychopharmacology 33(7):1570–1583
Naldini L, Blomer U, Gage FH, Trono D, Verma IM (1996) Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A 93(21):11382–11388
Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L (2000) Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 25(2):217–222
Acknowledgments
This work was supported by the Michael J Fox Foundation for Parkinson’s Research and the Parkinson’s UK as well as by the Italian Ministry of Health, the Fondazione CARIPLO and the Compagnia di San Paolo (to RB). Daniel Orellana conducted this study as partial fulfillment of his PhD in Molecular Medicine, Program in Neuroscience, Vita-Salute San Raffaele University, Milan, Italy. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory established by the San Raffaele Scientific Institute and the Vita-Salute San Raffele University.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Orellana, D., Morella, I., Indrigo, M., Papale, A., Brambilla, R. (2012). The Extracellular Signal-Regulated Kinase (ERK) Cascade in Neuronal Cell Signaling. In: Mukai, H. (eds) Protein Kinase Technologies. Neuromethods, vol 68. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-824-5_8
Download citation
DOI: https://doi.org/10.1007/978-1-61779-824-5_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-823-8
Online ISBN: 978-1-61779-824-5
eBook Packages: Springer Protocols