ERK-MAP Kinase Signaling in the Cytoplasm
ERK-MAPK is activated by dual phosphorylation of its activation loop TEY motif by the MEK-MAPKK. ERK cytoplasmic activity should be measured by assaying both the level of dually phosphorylated ERK and the level of phosphorylated substrate. We describe two
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1. Introduction The extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPKs, ERK-MAPKs) localize to many cellular subcompartments, including the cytoplasm, cell membrane, Golgi apparatus, endosomes, and cytoskeleton. Upon dual phosphory‑ lation and activation by MAPK/ERK-activating kinase (MEK), a detectable fraction of ERK translocates to the nucleus (1–3). Activated ERK is also retained in the extranuclear subcompartments, where it acts upon cytoplasmic substrates (4–7). ERK localization and signaling specificity are controlled by scaffolds, anchoring proteins,
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_11, © Springer Science+Business Media, LLC 2010
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interaction domains, and dimerization (8–10). For example, ERK activity toward the cytoplasmic substrates p90 ribosomal S6 kinase (RSK) and cPLA2 requires unique scaffolding proteins and dimerization. Scaffolds and dimerization appear to be disposable for nuclear activity (11). The availability of antibodies that recognize dually phosphorylated ERK has led the signaling community to adopt the detection of phospho-ERK1/2 as a readout of ERK activity. However, the presence of phosphorylated ERK does not necessarily indicate its activity toward a specific cytoplasmic substrate. We describe two methods for quantitatively measuring ERK activity toward cytoplasmic RSK. There are four RSK isoforms, namely RSK 1–4. Each RSK isoform has two kinase domains, a C-terminal kinase domain that autophosphorylates RSK and an N-terminal kinase domain that phosphorylates RSK substrates. ERK phosphorylates RSK’s C-terminal kinase domain on T573 (numbering refers to human RSK1). The activated C-terminal kinase domain then autophosphorylates RSK at the hydrophobic motif S380. Phospho-S380 recruits PDK1, which phosphorylates RSK’s N-terminal kinase domain, leading to full RSK activation (12). Inactive RSK1/2 shuttles between the cytoplasm and nucleus but is likely activated in the cytoplasm by cytoplasmic ERK (7, 13). Like ERK, RSK phosphorylates both cytoplasmic and nuclear targets, and ERK-MAPK pathway activation leads to increased RSK translocation and/or retention in the nucleus (1, 14). We describe two methods to quantitatively measure ERK activity toward RSK in growth factor-stimulated cells. These methods can be easily modified to monitor ERK activity on other cytoplasmic targets, such as cPLA2, paxillin, and others (5, 11). The first method, immunoblotting for endogenous ERK and RSK phospho-epitopes, assesses ERK activation under native conditions. Since RSK is a cytoplasmic target of active ERK (7), this provides a simple assay for monitoring cytoplasmic ERK activation. However, one must be cautious when interpreting data from this assay since it does not directly test whether the observed RSK phosphorylation is due to ERK. In addition, RSK phosphorylation is maintained by multiple signaling pathways at later time points. In
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