👤 Stephen M Keyse

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRAS Show less

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of β6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10 Show less

Dual-specificity MAP kinase (MAPK) phosphatases (MKPs or DUSPs) are well-established negative regulators of MAPK signalling in mammalian cells and tissues. By virtue of their differential subcellular localisation and ability to specifically recognise, dephosphorylate and inactivate different MAPK isoforms, they are key spatiotemporal regulators of pathway activity. Furthermore, as they are transcriptionally regulated as downstream targets of MAPK signalling they can either act as classical negative feedback regulators or mediate cross talk between distinct MAPK pathways. Because MAPKs and particularly Ras/ERK signalling are implicated in cancer initiation and development, the observation that MKPs are abnormally regulated in human tumours has been interpreted as evidence that these enzymes can either suppress or promote carcinogenesis. However, definitive evidence of such roles has been lacking. Here we review recent work based on the use of mouse models, biochemical studies and clinical data that demonstrate key roles for MKPs in modulating the oncogenic potential of Ras/ERK signalling and also indicate that these enzymes may play a role in the response of tumours to certain anticancer drugs. Overall, this work reinforces the importance of negative regulatory mechanisms in modulating the activity of oncogenic MAPK signalling and indicates that MKPs may provide novel targets for therapeutic intervention in cancer. Show less

Dual-specificity MAP kinase phosphatases (MKPs) provide a complex negative regulatory network that acts to shape the duration, magnitude and spatiotemporal profile of MAP kinase activities in response to both physiological and pathological stimuli. Individual MKPs may exhibit either exquisite specificity towards a single mitogen-activated protein kinase (MAPK) isoform or be able to regulate multiple MAPK pathways in a single cell or tissue. They can act as negative feedback regulators of MAPK activity, but can also provide mechanisms of crosstalk between distinct MAPK pathways and between MAPK signalling and other intracellular signalling modules. In this review, we explore the current state of knowledge with respect to the regulation of MKP expression levels and activities, the mechanisms by which individual MKPs recognize and interact with different MAPK isoforms and their role in the spatiotemporal regulation of MAPK signalling. Show less

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. Show less

There are ten mitogen-activated protein kinase (MAPK) phosphatases (MKPs) that act as negative regulators of MAPK activity in mammalian cells and these can be subdivided into three groups. The first comprises DUSP1/MKP-1, DUSP2/PAC1, DUSP4/MKP-2 and DUSP5/hVH-3, which are inducible nuclear phosphatases. With the exception of DUSP5, these MKPs display a rather broad specificity for inactivation of the ERK, p38 and JNK MAP kinases. The second group contains three closely related ERK-specific and cytoplasmic MKPs encoded by DUSP6/MKP-3, DUSP7/MKP-X and DUSP9/MKP-4. The final group consists of three MKPs DUSP8/hVH-5, DUSP10/MKP-5 and DUSP16/MKP-7 all of which preferentially inactivate the stress-activated p38 and JNK MAP kinases. Abnormal MAPK signalling will have important consequences for processes critical to the development and progression of human cancer. In addition, MAPK signalling also plays a key role in determining the response of tumour cells to conventional cancer therapies. The emerging roles of the dual-specificity MKPs in the regulation of MAPK activities in normal tissues has highlighted the possible pathophysiological consequences of either loss (or gain) of function of these enzymes as part of the oncogenic process. This review summarises the current evidence implicating the dual-specificity MKPs in the initiation and development of cancer and also on the outcome of treatment. Show less

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling. Show less

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) are activated by dual threonine and tyrosine phosphorylation of a TEY motif. The highly related kinase ERK5 is also activated by phosphorylation at a TEY motif. Inactivation of ERK1/2 is achieved by distinct members of the dual-specificity protein phosphatase (DUSP) family, which are responsible for the specific, regulated de-phosphorylation of the TEY motif. These include both nuclear (DUSP5) and cytoplasmic (DUSP6) enzymes. DUSP6, a candidate tumour suppressor gene, is thought to be highly specific for inactivation of ERK1/2 but several reports have suggested that it may also inactivate ERK5. Here we have compared the ability of DUSP6 to regulate the ERK1/2 and ERK5 protein kinases. We find that DUSP6 binds to ERK1/2 in both yeast and human cells but fails to bind to ERK5. Recombinant ERK2 can induce catalytic activation of DUSP6 whereas ERK5 cannot. Ectopic expression of DUSP6 can de-phosphorylate a co-expressed ERK2 construct but does not de-phosphorylate ERK5. Finally, expression of DUSP6 blocks the MEK1-driven activation of GAL4-ELK1, an ERK1/2-regulated transcription factor, but fails to block the MEK5-driven activation of GAL4-MEF2D, an ERK5-regulated transcription factor. These results demonstrate that even upon over-expression DUSP6 fails to inactivate ERK5, confirming that it is indeed an ERK1/2-specific DUSP. Show less

The regulated dephosphorylation of mitogen-activated protein kinases (MAPKs) plays a key role in determining the magnitude and duration of kinase activation and hence the physiological outcome of signalling. In mammalian cells, an important component of this control is mediated by the differential expression and activities of a family of 10 dual-specificity (Thr/Tyr) MAPK phosphatases (MKPs). These enzymes share a common structure in which MAPK substrate recognition is determined by sequences within an amino-terminal non-catalytic domain whereas MAPK binding often leads to a conformational change within the C-terminal catalytic domain resulting in increased enzyme activity. MKPs can either recognize and inactivate a single class of MAP kinase, as in the specific inactivation of extracellular signal regulated kinase (ERK) by the cytoplasmic phosphatase DUSP6/MKP-3 or can regulate more than one MAPK pathway as illustrated by the ability of DUSP1/MKP-1 to dephosphorylate ERK, c-Jun amino-terminal kinase and p38 in the cell nucleus. These properties, coupled with transcriptional regulation of MKP expression in response to stimuli that activate MAPK signalling, suggest a complex negative regulatory network in which individual MAPK activities can be subject to negative feedback control, but also raise the possibility that signalling through multiple MAPK pathways may be integrated at the level of regulation by MKPs. Show less

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity. Show less

MAP kinase phosphatase (MKP)-3 is a cytoplasmic dual specificity protein phosphatase that specifically binds to and inactivates the ERK1/2 MAP kinases in mammalian cells. However, the molecular basis of the cytoplasmic localization of MKP-3 or its physiological significance is unknown. We have used MKP-3-green fluorescent protein fusions in conjunction with leptomycin B to show that the cytoplasmic localization of MKP-3 is mediated by a chromosome region maintenance-1 (CRM1)-dependent nuclear export pathway. Furthermore, the nuclear translocation of MKP-3 seen in the presence of leptomycin B is mediated by an active process, indicating that MKP-3 shuttles between the nucleus and cytoplasm. The amino-terminal noncatalytic domain of MKP-3 is both necessary and sufficient for nuclear export of the phosphatase and contains a single functional leucine-rich nuclear export signal (NES). Even though this domain of the protein also mediates the binding of MKP-3 to MAP kinase, we show that mutations of the kinase interaction motif which abrogate ERK2 binding do not affect MKP-3 localization. Conversely, mutation of the NES does not affect either the binding or phosphatase activity of MKP-3 toward ERK2, indicating that the kinase interaction motif and NES function independently. Finally, we demonstrate that the ability of MKP-3 to cause the cytoplasmic retention of ERK2 requires both a functional kinase interaction motif and NES. We conclude that in addition to its established function in the regulated dephosphorylation and inactivation of MAP kinase, MKP-3 may also play a role in determining the subcellular localization of its substrate. Our results reinforce the idea that regulatory proteins such as MKP-3 may play a key role in the spatio-temporal regulation of MAP kinase activity. Show less

The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates. Show less

Mitogen-activated-protein kinase (MAP kinase) cascades are effector mechanisms for many growth factor signals implicated in developmental processes, including appendage outgrowth and organogenesis. The cascade culminates in extracellular-signal-regulated MAP kinase (ERK), which enters the nucleus. ERK activity reflects the competing actions of upstream activator kinases and inhibitory MAP kinase phosphatases. We have studied embryonic expression of the dual-specificity MAP kinase phosphatase PYST1/MKP3, which is a specific and potent regulator of the ERK class of MAP kinases. We found dynamic patterns of mPyst1 messenger RNA in important signalling centres associated with cell proliferation and patterning in developing mouse embryos, including presegmental paraxial mesoderm, limb bud and branchial arch mesenchyme, midbrain/hindbrain isthmus, and nasal, dental, hair, and mammary placodes. Most of these have been characterised as sites of FGF/FGFR signalling. Show less

Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as de-adhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesion-related substrates. Show less

Treatment of cells with cisplatin induces a sustained activation of the stress activated protein kinase SAPK/JNK and the mitogen-activated protein kinase p38. Activation of JNK by cisplatin is necessary for the induction of apoptosis. Expression of the MAPK phosphatases CL100/MKP-1 and hVH-5 selectively prevents JNK/SAPK activation by cisplatin in a dose dependent fashion and results in protection against cisplatin-induced apoptosis. In contrast, expression of the ERK-specific phosphatase Pyst1 inhibits JNK/SAPK activity only when expressed at very high levels and does not confer protection against cisplatin. Furthermore, expression of a catalytically inactive mutant of CL100 in 293 cells decreases the IC50 for cisplatin and increases the toxicity of transplatin. This effect seems to be mediated by an increase in JNK activity since p38 activity is unaffected. These results suggest that dual-specificity MAPK phosphatases may be candidate drug targets in order to optimize cisplatin based therapeutic protocols. Show less

Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen-induced gene expression and cell cycle re-entry. Show less

We have isolated the human genes encoding the Pyst1 (MKP-3) and Pyst2 (MKP-X) MAP kinase phosphatases. Both genes consist of three exons interrupted by two introns and lack an intron which is conserved in all the other members of this gene family characterised to date. This reinforces the conclusion that Pyst1 and Pyst2 are members of a distinct and structurally homologous subfamily of dual-specificity (Thr/Tyr) MAP kinase phosphatases. We find that Pyst2 mRNA is constitutively expressed in a wide variety of human cell lines including those derived from ovarian, bladder and breast cancers. While there is no evidence for inducible expression of Pyst2 mRNA in human skin fibroblasts in response to cellular stress, Pyst2 mRNA levels are moderately increased in response to serum stimulation. Pyst2 protein is predominantly cytosolic when expressed in COS-1 cells. In common with Pyst1, Pyst2 shows substrate selectivity for the classical p42 (ERK2) isoform of MAP kinase both in vitro and in vivo, displaying much reduced activity towards stress activated MAP kinase isoforms such as JNK-1 and p38/RK. Pyst2 binds p42 MAP kinase in vivo and both MAP kinase binding and substrate selectivity correlate with the ability of different recombinant MAP and SAP kinases to cause catalytic activation of the Pyst2 phosphatase in vitro. Show less

The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location. Show less