Studying the Regulation of MAP Kinase by MAP Kinase Phosphatases In Vitro and in Cell Systems
Signaling through MAPK pathways involves a network of activating kinases and inactivating phosphatases. While single MAPK kinases account for specific activation of the distinct MAPKs, inactivation of MAPKs by phosphatases involves a wider spectrum of enz
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ntroduction Inactivation of MAP kinase (MAPK) cascades by protein phosphatases is crucial to control the strength, duration, and location of MAPK signaling events (1). Specific serine/threonine phosphatases (PPs), tyrosine phosphatases (PTPs), and dual-specificity phosphatases (DSPs) exist that inactivate MAPKs by direct dephosphorylation of their TXY motifs. PPs that dephosphorylate MAPKs include several PP2A and PP2C enzymes (2, 3). However, the activities of PP2A and PP2C toward components of the MAPK pathways are not only restricted to MAPK Céline Tárrega and Caroline Nunes-Xavier contributed equally to this work.
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_18, © Springer Science+Business Media, LLC 2010
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ephosphorylation, but also target other upstream kinases. In d addition, both inhibitory and activating effects of these phosphatases on the MAPK pathways have been documented, depending on the PP2A holoenzyme, the PP2C isozyme, and the MAPK pathway (4, 5). PP2A forms functional complexes with ERK1/2 and the PTP HePTP (6), and the PP2C enzymes PP2Ca and Wip1 associate physically with p38 (7, 8), which could account for substrate specificity; however, the molecular basis of these associations are mostly unknown. In general, tyrosine and dual-specificity MAPK phosphatases are regarded as specific negative regulators of MAPK pathways by direct dephosphorylation and inactivation of the MAPKs. MAPK phosphatases of the tyrosine-specific classical PTP family are encoded by three genes in mammals, whose products are the non-nuclear proteins PTP-SL/PTPBR7, HePTP, and STEP. HePTP is mostly expressed in lymphoid tissues, whereas PTP-SL/PTPBR7 and STEP (both of which display multiple protein variants) are mostly expressed in the brain (9, 10). The MKP family of dual-specificity MAPK phosphatases, which is distinct from the classical PTP family, constitutes the major group of direct MAPK inactivating enzymes. There are ten MKP genes in mammals encoding active phosphatases, both nuclear and non-nuclear (MKP1, MKP2, DUSP2, DUSP5, MKP3, MKP4, MKPX, DUSP8, MKP7, and MKP5) (11, 12). The expression of several MKPs (MKP1, MKP3, DUSP2, and DUSP5) is induced as a consequence of the activation of the MAPK pathways, and serves as a negative feedback regulatory loop for these pathways (13, 14). Finally, a group of low molecular weight atypical DSPs (LMW-aDSPs) also dephosphorylate and inactivate MAPKs (15, 16). VHR, which dephosphorylates ERK1/2 and JNK, is the LMW-aDSP most extensively studied (17). For a more comprehensive list of PTP- and dual-specificity MAPK phosphatases, and for the standardized protein and gene names, see refs. 18, 19. MAPK-specific PTPs and MKPs possess a regulatory MAPK-binding domain that provides substrate specificity and promotes efficient dephosphorylation of MAPKs. Binding specificity is mainly achieved by the interaction between a docking motif in the MAPK-binding domain of the phospha
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