Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity protein phosphatase that can dephosphorylate and inactivate both mitogen-and stress-activated protein kinases in vitro and in vivo. However, the molecular mechanism responsible for the substrate selectivity of MKP-1 is unknown. In addition, it has been suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase substrate for MKP-1. We have used the yeast two-hybrid assay to demonstrate that MKP-1 is able to interact selectively with the extracellular signal-regulated kinase 1/2 (ERK1/2), p38␣, and c-Jun We conclude that the substrate selectivity of MKP-1 is determined by specific protein-protein interactions coupled with catalytic activation of the phosphatase and that these interactions are restricted to members of the MAP kinase family of enzymes.The mitogen-activated protein (MAP) 1 kinases are key components of cellular signal transduction pathways, which become activated in response to a wide variety of external stimuli. They can be subdivided into at least three classes based on sequence homology and differential activation by agonists (1-3); these include the growth factor-activated MAP kinases, extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the stress-activated MAP kinases c-Jun amino-terminal kinase (JNK, SAPK1) and p38 (SAPK2) MAP kinases. In addition, a number of less well characterized members of the MAP kinase family have been identified such as BMK1/ERK5, ERK7, and ERK3 (4 -6). MAP kinase pathways relay, amplify, and integrate complex signals in order to elicit appropriate biological responses. In mammalian cells, these include cellular proliferation, differentiation, inflammatory responses, and apoptosis. These responses are associated with significant alterations in the pattern of cellular gene expression, and transcription factors are a major target of MAP kinase signaling in vivo (7). To phosphorylate these proteins, activated MAP kinases translocate to the cell nucleus, a process that is generally associated with prolonged activation of the MAP kinase (8). Therefore, the magnitude and duration of MAP kinase activation are critical determinants of biological outcome, and regulatory mechanisms governing the activation of MAP kinase are of key importance in both physiological and pathological cell functions.The duration and magnitude of MAP kinase activation can be regulated at many points within the signal transduction pathway. However, it is now clear that the MAP kinase itself is a major target for regulation through the action of specific protein phosphatases. MAP kinase activation is dependent on the phosphorylation of both the threonine and tyrosine residues of the TXY motif found within the "activation loop" of the kinase. Since phosphorylation of both residues is required for activity, dephosphorylation of either residue is sufficient for inactivation. This can be achieved by protein-tyrosine ph...

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.

The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.The mammalian p38 mitogen-activated protein kinase (MAPK) pathway is activated by UV radiation, sodium arsenite, heat shock, bacterial lipopolysaccharide, and proinflammatory cytokines and is an important mediator of the cellular response to environmental stress (18,26,39). Among the cellular responses to p38 signaling are the production of inflammatory cytokines and phosphorylation of the small heat shock proteins. The physiological processes affected by p38 signaling include cell cycle progression, differentiation, apoptosis, and the inflammatory response. In addition, studies of Drosophila, Xenopus, and mouse have revealed important roles for p38 MAPK signaling during early development (32).Four distinct p38 MAPKs have been identified in mammalian cells: p38␣ (stress-activated protein kinase 2a [SAPK2a]), p38␤ (SAPK2b), p38␥ (SAPK3), and p38␦ (SAPK4). These enzymes are all phosphorylated and activated by MAPK kinase 6 (MKK6) (15). A second MKK, MKK3, is able to activate p38␣, p38␥, and p38␦, but not p38␤ (49). The four p38 MAPK isoforms are 60 to 70% identical at the amino acid sequence level and have overlapping substrate specificities in vitro. This has made the identification of physiological substrates and functions for p38 MAPKs a difficult task. However, the discovery that p38␣ and p38␤, but not p38␥ or 38␦, are inhibited by the pyridinyl imidazole SB203580 coupled with the more recent generation of mice which lack either individual p38 MAPKs or their upstream activators has proven extremely useful in delineating functional pathways for these enzymes (6,25,29,32) Targets of the p38 pathway inclu...

MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3.Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by ϳ50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.

Photodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericinmediated PDT of human cancer cells leads to upregulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE 2 . Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK ␣ and ␤ mediate COX-2 up-regulation at the protein and messenger levels. The p38 MAPK inhibitor, PD169316, abrogated COX-2 expression in PDT-treated cells, whereas overexpression of the drug-resistant PD169316-insensitive p38 MAPK ␣ and ␤ isoforms restored COX-2 levels in the presence of the kinase inhibitor. Transcriptional regulation by nuclear factor-B was not involved in COX-2 up-regulation by PDT. The half-life of the COX-2 messenger was drastically shortened by p38 MAPK inhibition in transcriptionally arrested cells, suggesting that p38 MAPK mainly acts by stabilizing the COX-2 transcript. Overexpression of WT-p38 MAPK increased cellular resistance to PDT-induced apoptosis, and inhibiting this pathway exacerbated cell death and prevented PGE 2 secretion. Hence, the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK may improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributes to tumor growth by the release of growthand pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis.