Reconstitution of the Nuclear Transport of the MAP Kinase ERK2
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
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1. Introduction 1.1. Nuclear– Cytoplasmic Distribution of ERK2
The mitogen-activated protein kinase (MAPK) signaling module containing the extracellular signal-regulated kinases 1/2 (ERK1/2) is subject to diverse modes of control within the cell (1). These include modulating activation kinetics of the enzyme, spatial restriction to distinct subcellular compartments and dynamic trafficking to sites of action. One such method of regulation is the nuclear translocation of ERK1/2, in response to various hormones or changes in cell state. Moreover, the mechanisms that dominate the control of ERK1/2 nuclear entry depend upon on cell type, context, activation state, and stimulus specificity. Unphosphorylated, phosphorylated, and active ERK1/2 have distinct but overlapping modes of nuclear entry. Initial studies
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_16, © Springer Science+Business Media, LLC 2010
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examining ERK1/2 localization suggested that nuclear translocation occurs upon stimulation with growth factors that cause the phosphorylation and activation of these protein kinases (2). Two groups have shown that unphosphorylated ERK2 can enter the nucleus independently from active import processes that require carrier proteins and the Ran-GTP cycle (3, 4). Instead, the nuclear entry of inactive ERK2 can occur through its direct interactions with the nucleoporins (Nups), proteins of the nuclear pore complex, in a manner independent of transport factors and Ran-GTP. Live-cell imaging studies provide some support for the energy-independent entry mechanism, as well as potentially contradictory views. One study suggested that ERK2 phosphorylation and activation are required for nuclear shuttling because inhibiting the upstream MAPK kinases MEK1/2 with U0126 blocked the nuclear accumulation of ERK2 (5). Fluorescence resonance energy transfer and recovery after photobleaching (FRET, FRAP) showed a steady-state rate of energyindependent entry of ERK2 even in unstimulated cells, confirmed the cytoplasmic association of ERK2 and MEK, and suggested that dissociation from MEK was sufficient to account for growth factor-enhanced nuclear entry (6). Because dissociation from MEK is caused by ERK2 activation, the MEK inhibitor will disfavor their dissociation; this suggests a possible reconciliation of the live-cell findings. Later reconstitution studies indicated that phosphorylated ERK2 can enter by both energy-independent and energy-dependent transport, as the addition of exogenous transport factors and an energy regenerating system enhanced nuclear import of the protein kinase (7). Recently, a Ser–Pro–Ser sequence was identified in ERK1/2 that is phosphorylated and required for importin 7-mediated nuclear translocation upon stimulation with serum or tissue plasminogen activator (8). Several other proteins have been implicated in the nuclear trafficking of ERK1/2. In addition to the direct facilitated o
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