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Protein phosphatases and the regulation of mitogen-activated protein kinase signalling
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Protein phosphatases and the regulation of mitogen-activated protein kinase signalling Stephen M Keyse The magnitude and duration of signalling through mitogen- and stress-activated kinases are critical determinants of biological effect. This reflects a balance between the activities of upstream activators and a complex regulatory network of protein phosphatases. These mitogen-activated protein kinase phosphatases include both dual-specificity (threonine/tyrosine) and tyrosine-specific enzymes, and recent evidence suggests that a single mitogen-activated protein kinase isoform may be acted upon by both classes of protein phosphatase. In both cases, substrate selectivity is determined by specific protein–protein interactions mediated through noncatalytic amino-terminal mitogen-activated protein kinase binding domains. Future challenges include the determination of exactly how this network of protein phosphatases interacts selectively with mitogen-activated protein kinase signalling complexes to achieve precise regulation of these key pathways in mammalian cells. Address Imperial Cancer Research Fund, Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital, Dundee DD1 9SY, Scotland, UK; e-mail: [email protected] Current Opinion in Cell Biology 2000, 12:186–192 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CD catalytic domain EGF epidermal growth factor ERK extracellular-signal-regulated kinase JNK c-jun amino-terminal kinase MAPK mitogen-activated protein kinase MKP MAPK phosphatase PTPase protein tyrosine phosphatase SAPK stress-activated protein kinase STEP striatal enriched phosphatase VHR vaccinia H1-related enzyme
Introduction The core module of a mitogen-activated protein kinase (MAPK) signalling pathway comprises a highly conserved cascade of three protein kinases. MAPKs are activated by phosphorylation of threonine and tyrosine residues within a signature sequence T–X–Y (single letter code) by a dual specificity MAPK kinase (MEK or MKK). These MKKs are in turn phosphorylated and activated by a diverse family of serine/threonine MKK kinases (MEKKs or MKKKs) [1]. MAPK pathways relay, amplify and integrate signals from a diverse range of stimuli and elicit an appropriate physiological response. In mammalian systems, these include cellular proliferation, differentiation, development, inflammatory responses and apoptosis. Thirteen mammalian MAPKs have been identified and classified on the basis of both sequence homology and
differential activation by agonists [2]. The first group includes the growth-factor-activated MAPKs ERK1 (extracellular-signal-regulated kinase) and ERK2 (MAPK1 and MAPK2), which contain the signature activation sequence T–E–Y. A second group of MAPKs are activated by cellular stress, including exposure to DNA damaging agents, oxidative stress, proinflammatory cytokines and protein synthesis inhibitors. For this reason, they are classified as stress-activated protein kinases (SAPKs). This group includes the c-jun amino-terminal kinases (JNK1–3 also known as SAPK1a, b and c) which contain the activation sequence T–P–Y and the p38 MAPK isoforms (SAPK2a and b, SAPK3 and SAPK4), all of which contain the activation sequence T–G–Y. The large number of MAPK components in mammalian cells presents a complex picture (Figure 1). However, although there are 14 or so MKKKs containing diverse regulatory domains, these feed into a more restricted network of only seven MKKs. The latter are highly specific for their MAPK substrates. Furthermore, the ability of different MAPK pathways to be selectively regulated is achieved, in part, by their association with scaffolding and anchoring proteins. These serve to tether the components of a particular MAPK module in a way that it can respond selectively to upstream inputs [3•]. Transcription factors are major targets for MAPKs and SAPKs. In order to phosphorylate these proteins, MAPKs must translocate from the cytoplasm to the nucleus. Translocation is generally associated with prolonged activation of MAPK. Therefore, both the magnitude and duration of MAPK activation are critical determinants of physiological outcome. The experimental system that best illustrates this is the differentiation of cultured rat PC12 cells. These cells proliferate in response to epidermal growth factor (EGF), while exposure to nerve growth factor (NGF) causes cell differentiation marked by neurite outgrowth. This differential response is entirely governed by the ability of NGF, but not EGF, to cause sustained activation and nuclear translocation of MAPK [4]. The duration and magnitude of MAPK activation may be regulated at many points within the signalling pathway. It is clear, however, that a major point of regulation occurs at the level of the MAPK. The activity of MAPK reflects a balance between the activities of the upstream activating kinase and protein phosphatases. Since phosphorylation of both threonine and tyrosine residues is required for activity, dephosphorylation of either is sufficient for inactivation. This can be achieved by tyrosine-specific phosphatases, serine/threonine-specific phosphatases or by dual specificity (threonine/tyrosine) protein phosphatases. This
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Figure 1 MAPK and SAPK pathways in mammalian cells. The MAPK module comprises an MKKK, and MKK (or MEK) and a MAPK. These pathways respond to extracellular signals, including growth and differentiation factors, cellular stress and cytokines. Once activated, MAPKs and SAPKs can phosphorylate a wide variety of proteins. These include transcription factors and other kinases (MAPKAP kinases). These downstream targets then control cellular responses including growth, differentiation, development and apoptosis [1,2,3•]. See text for full details.
Oxidative stress/ growth factors ?
Growth/neurotrophic factors/stress
Cytokines/ stress
RAS P
P GRB
? PAK ASK1 Tpl-2 MEKKs
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?
?
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review describes recent work that has revealed considerable complexity in the regulation of MAP kinase activity by these enzymes in mammalian cells. This includes the demonstration that both dual-specificity and tyrosinespecific phosphatases may regulate MAPKs in vivo and new insights into the way in which both of these classes of enzyme specifically recognise and dephosphorylate different MAP and SAP kinase isoforms.
Dual-specificity mitogen-activated protein kinase phosphatases Nine members of this family of MAPK phoshatases (MKPs) have been isolated and characterised in mammalian cells (Table 1). They all share a common structure, comprising a catalytic domain with significant amino acid sequence homology to a dual specificity PTPase (VH-1) from vaccinia virus and an aminoterminal noncatalytic domain containing two short regions of sequence homology with the catalytic domain of the cdc25 phosphatase [5,6]. These enzymes can be divided roughly into two groups, the inducible nuclear enzymes typified by CL100 (MKP-1) and a second group which includes Pyst1 (MKP-3). The latter group localise predominantly in the cytosol and are not encoded by immediate early genes. These enzymes are able to dephosphorylate both the threonine and tyrosine residues on ERK2 MAPK both in vitro and in vivo. This, coupled with the observation that many of these proteins are inducible by stimuli that activate MAPK pathways, strongly suggests that they participate in negative feedback control of MAPK activity [7–9].
RAF
Growth differentiation
p38/ SAPK2
Cytokine synthesis Growth apoptosis differentiation survival/apoptosis
Dual-specificity MKPs are able to selectively target different MAPK isoforms An important advance in our understanding of how this large family of proteins might act to regulate MAPK and SAPK signalling in mammalian cells came with the observation that certain MKPs display marked substrate selectivity for different MAPKs in vitro and in vivo. Recombinant Pyst1 (MKP-3) was found to be approximately 100-fold more active towards ERK2 than towards p38 (SAPK2) [10]. Furthermore, this substrate selectivity was also observed in vivo. M3/6 (hVH-5), another distinct cytosolic dual specificity MKP, has a reciprocal substrate selectivity when compared with Pyst1. It readily dephosphorylates and inactivates the SAPKs JNK1 and p38, but is without activity towards ERK2 [11].
Substrate binding causes catalytic activation of dual-specificity MKPs The dephosphorylation of ERK2 MAPK by Pyst1 (MKP3) in mammalian cells is accompanied by the formation of a tight physical complex between the phosphatase and ERK2 [10]. ERK2 binding is mediated by the aminoterminal noncatalytic domain of Pyst1 and loss of this domain abrogates substrate selectivity in vivo [12•]. In a surprising and exciting development, it was shown that ERK2 binding is accompanied by catalytic activation of the phosphatase in vitro as revealed by a greatly increased ability to hydrolyse the chromogenic substrate para-nitrophenylphosphate (p-NPP) [13••]. Catalytic activation mirrored the substrate selectivity of Pyst1, as SAPKs were unable either to bind or increase catalytic activity. The closely related enzymes Pyst2 and MKP-4 also undergo
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Table 1 The mammalian MAP kinase phosphatases. Dual-specificity MKPs CL100/MKP-1 PAC-1 hVH2/MKP-2/Typ-1 hVH3/B23
Nuclear enzymes, encoded by genes that are either growth factor or stress inducible.
hVH5/M3/6 Pyst1/MKP-3/rVH6 Pyst2/MKPX MKP-4 MKP-5
Proteins are localised predominantly in the cytosol when expressed in mammalian cells. Not encoded by immediate early genes.
VHR
Dual specificity phosphatase, which is not part of the same gene family as the above. May target ERK1 and ERK2.
Tyrosine-specific MKPs PTP-SL/PTBR7/ PC12-PTP1/STEP
HePTP/LC-PTP
Neuronal PTPases. Both transmembrane and cytosolic forms are generated by alternative splicing. Target ERK1/2 and p38 but not JNK. Lymphoid-specific. Target ERK1/2 and p38 but not JNK.
Serine/threonine-specific phosphatases PP2A May target both MAP kinase and MAP kinase kinase. PP2Cα Seems to act on both p38 and JNK. Also able to inactivate MKKs.
catalytic activation on binding to ERK2 [13••,14]. These results indicate that this process may be a general mechanism by which all members of the dual specificity MKP family are regulated. However, MKP-5, a recently characterised MKP, both binds to and efficiently dephosphorylates p38 (SAPK2) but recombinant p38 MAPK does not activate MKP-5 in vitro [15]. How is this dramatic increase in Pyst1 activity achieved? Major clues have come with the determination of a crystal structure for the Pyst1 catalytic domain (Pyst1-CD) [16••]. Pyst1-CD adopts a typical protein tyrosine phosphatase (PTPase) fold with a shallow active site cleft. This latter feature, which is also found in the dual specificity phosphatase vaccinia H1-related enzyme (VHR), may explain the ability of these enzymes to accommodate both phosphotyrosine and phosphothreonine sidechains within the active site cleft [17]. The most striking feature of the Pyst1-CD structure, however, is the distorted geometry of key catalytic residues in the absence of substrate. Firstly, a highly conserved arginine residue (Arg299 in Pyst1) which in other PTPases co-ordinates the phosphate group of the substrate [18], is not well positioned for catalysis. Secondly, a highly conserved aspartic acid residue (Asp262 in Pyst1) is predicted to perform an essential catalytic role by acting as a general acid responsible for protonation of the tyrosine leaving group [18]. In Pyst1-CD Asp262 is displaced by almost 5.5 Å from the equivalent position in VHR,
indicating that it is unlikely to participate in catalysis. In agreement with this, mutation of Asp262 to asparagine (D262N, single letter amino acid code) in full length Pyst1 had no significant effect on the rate of p-NPP hydrolysis in the absence of ERK2. This contrasts with results obtained for both VHR and PTP1B, where substitution of this residue greatly decreases activity [19]. However, the D262N mutant of Pyst1 fails to undergo catalytic activation on addition of ERK2, indicating that this residue is only required for catalysis in the activated form of Pyst1 [16••]. In support of a role for catalytic activation in vivo, this mutant is also severely compromised in its ability to dephosphorylate ERK2 in mammalian cells. The simplest interpretation of these data is that Asp262 and its associated loop undergo closure over the active site of Pyst1 only when ERK2 is bound to the phosphatase, thus positioning the aspartate residue in an optimal conformation for catalysis. However, the precise nature of these conformational changes must await the determination of a structure of a co-complex of Pyst1 and ERK2.
Physiological roles for dual-specificity MKPs Despite the evidence in support of a role for these enzymes in regulating MAPKs and SAPKs in vivo, definitive proof is still lacking in mammalian systems. The mouse CL100 (MKP-1) gene has been disrupted and these animals develop normally and are fertile. Furthermore, cells cultured from these animals do not display any abnormalities in either MAPK activation or inactivation [20]. However, direct evidence of a role for these enzymes has come from genetic and biochemical studies in yeasts and Drosophila. The Msg5p enzyme of Saccharomyces cerevisiae is most similar in sequence to human CL100 (MKP-1), and was isolated as a suppressor of pheromone-induced G1 arrest, which is mediated by the Fus3p MAPK in haploid yeast cells [21]. Deletion of MSG5 results in increased activation of Fus3p, and Msg5p efficiently dephosphorylates both the threonine and tyrosine residues of Fus3p in vitro. The observation that MSG5 is pheromone inducible and that this induction is mediated by FUS3 indicates that this phosphatase is involved in negative feedback control of MAPK signalling. More recently, two further dual specificity MKPs have been identified in yeasts: CPP1, a gene which is highly related to MSG5, has been isolated from the human opportunistic pathogen Candida albicans and regulates a MAPK pathway involved in the yeast to hyphal transition [22]; the pmp1+ gene in Schizosaccharomyces pombe encodes a protein that is closely related to Msg5p and dephosphorylates the fission yeast MAPK Pmk1p in vitro and in vivo [23]. Pmk1p functions co-ordinately with a protein kinase C pathway and is implicated in the maintenance of cell integrity. This suggests that pmp1+, like MSG5, may be involved in feedback control of MAP kinase activity. It is not yet clear,
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Figure 2
Figure 3
(a)
(b) MEKK?
(a)
Raf
MKKK
Ssk1p
(b)
Ssk2p/ssk22p
hep
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puc
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PTP-ER Eye development
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MAPK signalling pathways in Drosophila are regulated by both dual specificity and PTPases. (a) During embryogenesis, the Drosophila JNK pathway is activated in the cells at the leading edge of the epidermis. Djun is then activated and is involved in mediating expression of the dual specificity MKP encoded by puc, and also dpp (TGFβ). Puc then inactivates bsk (JNK) thus regulating its own expression [26••]. Dpp is involved in regulating cellular events including those required for dorsal closure. (b) The Drosophila MAPK pathway which lies downstream of Ras, and contains Raf and the rolled (rl) MAPK, acts as a binary switch during eye development. Signalling triggers the differentiation of only one of five identical cells as an R7 neuron, whereas the lack of signal transmission in the other four cells causes them to adopt the non-neuronal cone cell fate. PTPER has now been identified as a tyrosine-specific MKP which inactivates rl and plays a physiological role in regulating this signalling pathway [40••].
Both tyrosine-specific and dual specificity MKPs coordinately regulate a MAPK signalling pathway in S. cerevisiae. (a) The Hog1p osmoregulatory MAPK in budding yeast is regulated by two tyrosine specific phosphatases encoded by PTP2 and PTP3 [27,28]. Of these, Ptp2p is the most critical. (b) Ptp3p also plays a role in regulating the pheromone-responsive MAPK Fus3p where it acts in concert with the inducible dual-specificity MKP encoded by MSG5 to regulate the adaptive response to pheromone [29]. Positive interactions are indicated by arrows while negative regulation is indicated by bars. Different line weights indicate potency of activities. Dual-specificity MKPs are boxed, whereas tyrosine-specific MKPs are underlined.
able to inactivate JNK. Interestingly, both loss and overexpression of puc have adverse effects on dorsal closure, indicating that JNK activity must be tightly controlled within critical limits for normal development to occur. Finally, the expression of puc is dependent on the activity of either bsk or its upstream activator, indicating that puc functions as part of a negative feedback control (Figure 2a).
Tyrosine-specific phosphatases also regulate MAPK signalling however, whether either Pm1p protein levels or activity are modulated in response to stimuli that activate Pmk1p. Perhaps the most informative experiments providing direct proof of a physiological role for a dual-specificity phosphatase in the regulation of MAP kinase activity in higher eukaryotes have come from studies in Drosophila. Basket (bsk), the Drosophila homologue of the mammalian JNK (or SAPK1) plays a role in the process of dorsal closure during embryogenesis [24]. Dorsal closure is a process in which lateral epithelial cells undergo morphological changes and migrate to cover the dorsal region of the embryo. Ring and Martinez-Ariaz [25] identified mutations in the puckered (puc) gene as affecting this process and puc was subsequently found to encode a CL100-like dual specificity phosphatase [26••]. Biochemical assays showed that extracts from wild type but not puc mutant embryos were
A role for PTPases in regulating MAPK first came from genetic and biochemical studies of the osmoregulatory MAPK pathways in yeasts. In S. cerevisiae, the osmoticstress-responsive Hog1p MAPK is co-ordinately regulated by two PTPases encoded by PTP2 and PTP3 [27,28]. One unexpected twist in this story came with the finding that PTP3, together with the gene encoding the dual specificity phosphatase MSG5, also plays a role in the regulation of the pheromone-responsive Fus3p MAPK [29]. This was the first indication that a single MAP kinase could be regulated by more than one class of protein phosphatase (Figure 3). In S. pombe, the functional equivalent of the Hog1p MAPK Sty1/Spc1 is also regulated by two PTPases Pyp1 and Pyp2 [30,31]. Furthermore, Pyp2 levels are increased by activation of Sty1/Spc1. Thus, like the inducible dual specificity MKPs, these enzymes are implicated in feedback control of MAPK activity.
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Given the high level of conservation of MAPK pathways from yeasts to man, it would be surprising if mammalian equivalents of these tyrosine-specific MKPs did not exist. Recent work has now led to the discovery of three gene families of closely related PTPases which regulate MAPK signalling in mammalian cells. STEP (striatal enriched phosphatase) and PTP-SL (STEP-like phosphatase), PTPases which are expressed in neuronal cells, were found to physically associate with ERK1 and ERK2 [32•]. On binding to ERKs, two distinct events occurred: firstly, the noncatalytic amino termini of both PTP-SL and STEP were phosphorylated; secondly, the MAPK was dephosphorylated and inactivated, specifically on the tyrosine residue within the regulatory T–E–Y motif. A 16-aminoacid motif within the noncatalytic amino-terminal domain of both PTPases was identified as being critical for ERK binding and dephosphorylation and was termed the kinase interaction motif (KIM). Expression of PTP-SL was also able to suppress ERK activation in response to EGF in Cos-7 cells [32•]. Similar results were obtained for the phosphatase PTPBR7, an enzyme which is closely related to PTP-SL and STEP [33]. Interestingly, both cytosolic and transmembrane isoforms of PTP-SL were able to downregulate ERK2 activity indicating that these enzymes might act on distinct pools of ERK2 within the cell.
What physiological role do these tyrosine-specific enzymes play? HePTP is able to suppress T cell receptor mediated activation and may modulate cytokine production [34,35•,36•], and the neuronal PTPase PC12-PTP1 (a rat homologue of PTPBR7) is inducible by NGF, indicating that it may play a role in modulating MAPK activity in response to growth and differentiation factors [39]. However, direct in vivo evidence for a physiological role of a PTP in regulating ERK/MAPK in higher eukaryotes has again come from genetic studies of MAPK signalling in Drosphila.
The related lymphoid-specific PTPases, haemopoietic PTP (HePTP) and leukocyte PTP (LC-PTP), were found to block antigen-dependent T-lymphocyte activation [34,35•,36•], and cellular targets of HePTP/LC-PTP were identified as ERK1 and ERK2. Like PTP-SL and STEP, HePTP binds to ERK2 through its noncatalytic amino-terminal domain, which also contains a KIM motif. HePTP was also able to bind p38 MAPK (SAPK2) but was unable either to form complexes with JNK (SAPK1), or to inhibit JNK activity [35•]. Similar results have been reported for PTP-SL [37], reinforcing the notion that, just as for the dual specificity MKPs, substrate selectivity is determined by specific protein–protein interactions.
Conclusions and future perspectives
Like both PTP-SL and STEP, the amino-terminal domain of HePTP is also phosphorylated by ERKs. Two sites of modification, Thr45 and Ser72, have been identified [35•]. Interestingly, mutation of these residues to alanine augments the activity of HePTP, indicating that phosphorylation might play a role in dissociating ERK and HePTP [35•]. This idea is lent further support by the recent finding that Ser23 in HePTP, which lies within the minimal KIM motif, is phosphorylated by cAMP-dependent protein kinase (PKA) in vitro and in vivo [38•]. Furthermore, modification of this residue reduces the binding of ERK2 to HePTP and this dissociation is associated with increased ERK2 activity. Thus, it appears that phosphorylation of HePTP by both MAPKs and PKA may modify the ability of the phosphatase to bind to its substrate. In the latter case, this may be an important point of cross-talk between the MAPK and cAMP signalling pathways.
The Drosophila rolled (rl) MAPK gene, which encodes a protein most similar to mammalian ERK1 and 2, is implicated in signalling downstream of multiple receptor tyrosine kinases. This MAPK pathway, which lies downstream of Ras1, acts as a binary switch to trigger cell differentiation during eye development. Karim and Rubin [40••] carried out a genetic screen for mutations which act negatively downstream of Ras1 and isolated a PTP, protein tyrosine phosphatase-ERK/enhancer of Ras1 (PTP-ER). PTP-ER targets rl and is most similar in amino acid sequence to PTP-SL, STEP and HePTP. Furthermore, a KIM motif is located within the amino-terminal noncatalytic domain of the protein, and PTP-ER binds to and inactivates MAPK both in vitro and in vivo (Figure 2b).
A large number of mammalian MKPs have now been identified. These include both dual specificity and tyrosine-specific enzymes (Table 1). More may yet be discovered. The dual specificity phosphatase VHR, which lacks the amino-terminal domain found in other dual specificity MKPs, has recently been reported to target ERK2 [41] and there is evidence indicating that serine/threonine-specific phosphatases may also regulate MAPKs [42,43]. Specific protein–protein interactions mediated by the noncatalytic amino-terminal domains of both classes of MKPs are critical for substrate specificity. In the case of both Pyst1 and PTP-SL, these are sufficiently robust to anchor ERK2 protein in the cytoplasm of mammalian cells [37,44]. Interestingly, although there is no discernable amino acid homology between the MAPK-binding domains of mammalian dual specificity and tyrosine-specific MKPs, recent evidence suggests that they may recognise their MAPK targets in a similar way. The sevenmaker mutant of ERK2 (D321N), a gain-of-function mutant first identified in Drosophila [45], is known to be resistant to inactivation by dual specificity MKPs [46,47]. In the case of Pyst1 (MKP-3), ERK2 (D321N) is unable to bind the amino-terminal domain of the phosphatase and cause catalytic activation [20]. Very recently, it has been shown that both human HePTP [36•] and Drosophila PTPER [40••] are also unable to form complexes with, or inactivate, this ERK2 mutant. It will be interesting to determine the relative affinities of these interactions,
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particularly where a dual specificity MKP and a tyrosinespecific enzyme target the same MAPK isoform. Finally, MAPK modules, in common with other signalling pathways are regulated by organisation into higher order complexes [3•]. These complexes, which include both upstream activators and also noncatalytic proteins such as scaffolds and inhibitory proteins, will determine where and when a particular pathway is active. We will need to understand how the MKPs are integrated into these signalling assemblies before we can fully understand how these enzymes participate in the overall control of these key pathways.
Update Since the submission of this review, two important papers have been published that are particularly relevant to this article [48•,49•].
Acknowledgements The author acknowledges the support of the Imperial Cancer Research Fund and would also like to thank David Slack and Ole-Morten Seternes for critical reading of the manuscript.
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kinase by the PTP2 and PTP3 protein-tyrosine phosphatases. Mol Cell Biol 1997,17:1289-1297. 28. Jacoby T, Flanagan H, Faykin A, Seto AG, Mattison C, Ota I: 2 protein-tyrosine phosphatases inactivate the osmotic-stress response pathway in yeast by targeting the mitogen-activated protein kinase, Hog1. J Biol Chem 1997, 272:17749-17755. 29. Zhan XL, Deschenes RJ, Guan KL: Differential regulation of FUS3 map kinase by tyrosine-specific phosphatases PTP2/PTP3 and dual-specificity phosphatase Msg5 in Saccharomyces-cerevisiae. Genes Dev 1997, 11:1690-1702.
39. Sharma E, Lombroso PJ: A neuronal protein tyrosine phosphatase induced by nerve growth factor. Proc Natl Acad Sci USA 1995, 270:49-53. 40. Karim FD, Rubin GM: PTP-ER, a novel tyrosine phospatase, •• functions downstream of Ras1 to downregulate MAP kinase during Drosophila eye development. Mol Cell 1999, 3:741-750. A genetic screen is used to isolate a novel protein tyrosine phosphatase PTP-ER, which dephosphorylates and inactivates MAP kinase in vitro and in vivo. This study provides direct in vivo evidence for a tyrosine-specific phosphatase regulating ERK/MAPK in higher eukaroytes.
30. Millar JBA, Buck V, Wilkinson MG: Pyp1 and pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cellsize at division in fission yeast. Genes Dev 1995, 9:2117-2130.
41. Todd JL, Tanner KG, Denu JM: Extracellular regulated kinases (ERK)1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. J Biol Chem 1999, 274:13271-13280.
31. Shiozaki K, Russell P: Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature 1995, 378:739-743.
42. Alessi DR, Gomez N, Moorhead G, Lewis T, Keyse SM, Cohen P: Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100 in various cell lines. Curr Biol 1995, 5:283-295.
32 •
Pulido R, Zuniga A, Ullrich A: PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signalregulated kinases ERK1 and ERK2 by association through a kinase interaction motif. EMBO J 1998, 17:7337-7350. This paper identifies a new class of tyrosine-specific phosphatases which regulate MAPK in mammalian cells. It also defines an interaction motif within the amini-terminal noncatalytic domain of these enzymes, which mediates ERK binding.
α inhibits 43. Takekawa T, Maeda T, Saito H: Protein phosphatase 2Cα the human stress-responsive p38 and JNK MAPK pathways. EMBO J 1998, 17:4744-4752.
33
45. Brunner D, Oellers N, Szabad J, Briggs WH, Zipursky SL, Hafen E: A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase pathways. Cell 1994, 76:875-888.
Ogata M, Oh-hora M, Kosugi A, Hamaoka T: Inactivation of mitogenactivated protein kinases by a mammalian tyrosine-specific enzyme. Biochem Biophys Res Commun 1999, 256:52-55.
44. Brunet A, Roux D, Lenormand P, Dowd S, Keyse SM, Pouyssegur J: Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 1999, 18:664-674.
34. Saxena M, Williams S, Gilman J, Mustelin T: Negative regulation of T cell antigen receptor signal transduction by hematopoietic tyrosine phosphatase (HePTP). J Biol Chem 1998, 273:15340-15344.
46. Bott CM, Thorneycroft SG, Marshall CJ: The sevenmaker gain-offunction mutation in p42 MAP kinase leads to enhanced signalling and reduced sensitivity to dual-specificity phosphatase action. FEBS Lett 1995, 352:201-205.
35. Saxena M, Williams S, Brockdorff J, Gilman J, Mustelin T: Inhibition of • T cell signalling by mitogen-activated protein kinase-targeted hematopoietic tyrosine phosphatase (HePTP). J Biol Chem 1999, 274: 11693-11700. This paper identifies the target for HePTP in mediating suppression of antigen receptor-induced transcription from the interleukin gene promoter as ERK1 and ERK2.
47.
36. Oh-hora M, Ogata M, Mori Y, Adachi M, Imai K, Kosugi A, Hamaoka T: • Direct suppression of TCR-mediated activation of extracellular signal-regulated kinase by leukocyte protein tyrosine phosphatase, a tyrosine-specific phosphatase. J Immunol 1999, 163:1282-1288. This paper identifies ERK1 and ERK2 as substrates for leukocyte PTP but also shows that this phospatase is unable to interact with or inactivate the sevenmaker ERK2 gain-of-function mutant. 37.
Zuniga A, Torres J, Ubeda J, Pulido R: Interaction of mitogenactivated protein kinases with the kinase interaction motif of the tyrosine phosphatase PTP-SL provides substrate specificity and retains ERK2 in the cytoplasm. J Biol Chem 1999, 274:21900-21907.
38. Saxena M, Williams S, Tasken K, Mustelin T: Crosstalk between • cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase. Nat Cell Biol 1999, 1:305-311. This paper indicates that crosstalk between the cAMP and MAPK signaling pathway may be mediated via phosphorylation of HePTP by protein kinase A, leading to dissociation of the complex between HePTP and its MAPK substrate.
Chu Y, Solski PA, Khosravi-Far R, Der CJ, Kelly K:The mitogenactivated protein kinase phosphatases PAC1, MKP-1 and MKP-2 have unique substrate specificities and reduced activity in vivo towards the ERK2 sevenmaker mutation. J Biol Chem 1996, 271:6497-6501.
48. Brondello J-M, Pouyssegur J, McKenzie FR: Reduced MAP kinase • phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 1999, 286:2514-2517. This paper shows that the inducible nuclear MKP-1(CL100) phosphatase is a labile protein and is targeted for degradation by the ubiquitin-directed proteasome complex. Furthermore, phosphorylation of MKP-1 on two carboxyterminal serine residues by p42MAPK or p44MAPK led to stabilisation of the protein. These results reveal a new facet of a complex control mechanism which is designed to prevent the undesirable effects of long-term activation of these key mitogenic signalling pathways. 49. Zhan X-L, Guan KL: A specific protein–protein interaction accounts • for the in vivo substrate selectivity of Ptp3 towards the Fus3 MAP kinase. Genes Dev 1999, 13:2811-2827. Tyrosine specific MKPs are found in both S. cerevisiae and mammalian cells. However, the kinase interaction motif (KIM) identified in the non-catalytic amino terminus of the mammalian enzymes, which is responsible for MAPK binding, is not present in yeast phosphatases such as Ptp3p which targets the Fus3p MAP kinase. Here the authors demonstrate that Ptp3p does bind to its substrate Fus3p via its non-catalytic amino-terminal domain. However, the sequence motif responsible is more similar to those identified in the noncatalytic amino terminal domains of the mammalian dual-specificity MKPs. This paper reinforces the concept that the specificity of MAP kinase inactivation in vivo is governed by specific protein–protein interactions with the phiosphatase catalytic domain.
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Protein phosphatases and the regulation of mitogen-activated protein kinase signalling Stephen M Keyse The magnitude and duration of signalling through mitogen- and stress-activated kinases are critical determinants of biological effect. This reflects a balance between the activities of upstream activators and a complex regulatory network of protein phosphatases. These mitogen-activated protein kinase phosphatases include both dual-specificity (threonine/tyrosine) and tyrosine-specific enzymes, and recent evidence suggests that a single mitogen-activated protein kinase isoform may be acted upon by both classes of protein phosphatase. In both cases, substrate selectivity is determined by specific protein–protein interactions mediated through noncatalytic amino-terminal mitogen-activated protein kinase binding domains. Future challenges include the determination of exactly how this network of protein phosphatases interacts selectively with mitogen-activated protein kinase signalling complexes to achieve precise regulation of these key pathways in mammalian cells. Address Imperial Cancer Research Fund, Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital, Dundee DD1 9SY, Scotland, UK; e-mail: [email protected] Current Opinion in Cell Biology 2000, 12:186–192 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CD catalytic domain EGF epidermal growth factor ERK extracellular-signal-regulated kinase JNK c-jun amino-terminal kinase MAPK mitogen-activated protein kinase MKP MAPK phosphatase PTPase protein tyrosine phosphatase SAPK stress-activated protein kinase STEP striatal enriched phosphatase VHR vaccinia H1-related enzyme
Introduction The core module of a mitogen-activated protein kinase (MAPK) signalling pathway comprises a highly conserved cascade of three protein kinases. MAPKs are activated by phosphorylation of threonine and tyrosine residues within a signature sequence T–X–Y (single letter code) by a dual specificity MAPK kinase (MEK or MKK). These MKKs are in turn phosphorylated and activated by a diverse family of serine/threonine MKK kinases (MEKKs or MKKKs) [1]. MAPK pathways relay, amplify and integrate signals from a diverse range of stimuli and elicit an appropriate physiological response. In mammalian systems, these include cellular proliferation, differentiation, development, inflammatory responses and apoptosis. Thirteen mammalian MAPKs have been identified and classified on the basis of both sequence homology and
differential activation by agonists [2]. The first group includes the growth-factor-activated MAPKs ERK1 (extracellular-signal-regulated kinase) and ERK2 (MAPK1 and MAPK2), which contain the signature activation sequence T–E–Y. A second group of MAPKs are activated by cellular stress, including exposure to DNA damaging agents, oxidative stress, proinflammatory cytokines and protein synthesis inhibitors. For this reason, they are classified as stress-activated protein kinases (SAPKs). This group includes the c-jun amino-terminal kinases (JNK1–3 also known as SAPK1a, b and c) which contain the activation sequence T–P–Y and the p38 MAPK isoforms (SAPK2a and b, SAPK3 and SAPK4), all of which contain the activation sequence T–G–Y. The large number of MAPK components in mammalian cells presents a complex picture (Figure 1). However, although there are 14 or so MKKKs containing diverse regulatory domains, these feed into a more restricted network of only seven MKKs. The latter are highly specific for their MAPK substrates. Furthermore, the ability of different MAPK pathways to be selectively regulated is achieved, in part, by their association with scaffolding and anchoring proteins. These serve to tether the components of a particular MAPK module in a way that it can respond selectively to upstream inputs [3•]. Transcription factors are major targets for MAPKs and SAPKs. In order to phosphorylate these proteins, MAPKs must translocate from the cytoplasm to the nucleus. Translocation is generally associated with prolonged activation of MAPK. Therefore, both the magnitude and duration of MAPK activation are critical determinants of physiological outcome. The experimental system that best illustrates this is the differentiation of cultured rat PC12 cells. These cells proliferate in response to epidermal growth factor (EGF), while exposure to nerve growth factor (NGF) causes cell differentiation marked by neurite outgrowth. This differential response is entirely governed by the ability of NGF, but not EGF, to cause sustained activation and nuclear translocation of MAPK [4]. The duration and magnitude of MAPK activation may be regulated at many points within the signalling pathway. It is clear, however, that a major point of regulation occurs at the level of the MAPK. The activity of MAPK reflects a balance between the activities of the upstream activating kinase and protein phosphatases. Since phosphorylation of both threonine and tyrosine residues is required for activity, dephosphorylation of either is sufficient for inactivation. This can be achieved by tyrosine-specific phosphatases, serine/threonine-specific phosphatases or by dual specificity (threonine/tyrosine) protein phosphatases. This
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Figure 1 MAPK and SAPK pathways in mammalian cells. The MAPK module comprises an MKKK, and MKK (or MEK) and a MAPK. These pathways respond to extracellular signals, including growth and differentiation factors, cellular stress and cytokines. Once activated, MAPKs and SAPKs can phosphorylate a wide variety of proteins. These include transcription factors and other kinases (MAPKAP kinases). These downstream targets then control cellular responses including growth, differentiation, development and apoptosis [1,2,3•]. See text for full details.
Oxidative stress/ growth factors ?
Growth/neurotrophic factors/stress
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RAS P
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? PAK ASK1 Tpl-2 MEKKs
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review describes recent work that has revealed considerable complexity in the regulation of MAP kinase activity by these enzymes in mammalian cells. This includes the demonstration that both dual-specificity and tyrosinespecific phosphatases may regulate MAPKs in vivo and new insights into the way in which both of these classes of enzyme specifically recognise and dephosphorylate different MAP and SAP kinase isoforms.
Dual-specificity mitogen-activated protein kinase phosphatases Nine members of this family of MAPK phoshatases (MKPs) have been isolated and characterised in mammalian cells (Table 1). They all share a common structure, comprising a catalytic domain with significant amino acid sequence homology to a dual specificity PTPase (VH-1) from vaccinia virus and an aminoterminal noncatalytic domain containing two short regions of sequence homology with the catalytic domain of the cdc25 phosphatase [5,6]. These enzymes can be divided roughly into two groups, the inducible nuclear enzymes typified by CL100 (MKP-1) and a second group which includes Pyst1 (MKP-3). The latter group localise predominantly in the cytosol and are not encoded by immediate early genes. These enzymes are able to dephosphorylate both the threonine and tyrosine residues on ERK2 MAPK both in vitro and in vivo. This, coupled with the observation that many of these proteins are inducible by stimuli that activate MAPK pathways, strongly suggests that they participate in negative feedback control of MAPK activity [7–9].
RAF
Growth differentiation
p38/ SAPK2
Cytokine synthesis Growth apoptosis differentiation survival/apoptosis
Dual-specificity MKPs are able to selectively target different MAPK isoforms An important advance in our understanding of how this large family of proteins might act to regulate MAPK and SAPK signalling in mammalian cells came with the observation that certain MKPs display marked substrate selectivity for different MAPKs in vitro and in vivo. Recombinant Pyst1 (MKP-3) was found to be approximately 100-fold more active towards ERK2 than towards p38 (SAPK2) [10]. Furthermore, this substrate selectivity was also observed in vivo. M3/6 (hVH-5), another distinct cytosolic dual specificity MKP, has a reciprocal substrate selectivity when compared with Pyst1. It readily dephosphorylates and inactivates the SAPKs JNK1 and p38, but is without activity towards ERK2 [11].
Substrate binding causes catalytic activation of dual-specificity MKPs The dephosphorylation of ERK2 MAPK by Pyst1 (MKP3) in mammalian cells is accompanied by the formation of a tight physical complex between the phosphatase and ERK2 [10]. ERK2 binding is mediated by the aminoterminal noncatalytic domain of Pyst1 and loss of this domain abrogates substrate selectivity in vivo [12•]. In a surprising and exciting development, it was shown that ERK2 binding is accompanied by catalytic activation of the phosphatase in vitro as revealed by a greatly increased ability to hydrolyse the chromogenic substrate para-nitrophenylphosphate (p-NPP) [13••]. Catalytic activation mirrored the substrate selectivity of Pyst1, as SAPKs were unable either to bind or increase catalytic activity. The closely related enzymes Pyst2 and MKP-4 also undergo
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Table 1 The mammalian MAP kinase phosphatases. Dual-specificity MKPs CL100/MKP-1 PAC-1 hVH2/MKP-2/Typ-1 hVH3/B23
Nuclear enzymes, encoded by genes that are either growth factor or stress inducible.
hVH5/M3/6 Pyst1/MKP-3/rVH6 Pyst2/MKPX MKP-4 MKP-5
Proteins are localised predominantly in the cytosol when expressed in mammalian cells. Not encoded by immediate early genes.
VHR
Dual specificity phosphatase, which is not part of the same gene family as the above. May target ERK1 and ERK2.
Tyrosine-specific MKPs PTP-SL/PTBR7/ PC12-PTP1/STEP
HePTP/LC-PTP
Neuronal PTPases. Both transmembrane and cytosolic forms are generated by alternative splicing. Target ERK1/2 and p38 but not JNK. Lymphoid-specific. Target ERK1/2 and p38 but not JNK.
Serine/threonine-specific phosphatases PP2A May target both MAP kinase and MAP kinase kinase. PP2Cα Seems to act on both p38 and JNK. Also able to inactivate MKKs.
catalytic activation on binding to ERK2 [13••,14]. These results indicate that this process may be a general mechanism by which all members of the dual specificity MKP family are regulated. However, MKP-5, a recently characterised MKP, both binds to and efficiently dephosphorylates p38 (SAPK2) but recombinant p38 MAPK does not activate MKP-5 in vitro [15]. How is this dramatic increase in Pyst1 activity achieved? Major clues have come with the determination of a crystal structure for the Pyst1 catalytic domain (Pyst1-CD) [16••]. Pyst1-CD adopts a typical protein tyrosine phosphatase (PTPase) fold with a shallow active site cleft. This latter feature, which is also found in the dual specificity phosphatase vaccinia H1-related enzyme (VHR), may explain the ability of these enzymes to accommodate both phosphotyrosine and phosphothreonine sidechains within the active site cleft [17]. The most striking feature of the Pyst1-CD structure, however, is the distorted geometry of key catalytic residues in the absence of substrate. Firstly, a highly conserved arginine residue (Arg299 in Pyst1) which in other PTPases co-ordinates the phosphate group of the substrate [18], is not well positioned for catalysis. Secondly, a highly conserved aspartic acid residue (Asp262 in Pyst1) is predicted to perform an essential catalytic role by acting as a general acid responsible for protonation of the tyrosine leaving group [18]. In Pyst1-CD Asp262 is displaced by almost 5.5 Å from the equivalent position in VHR,
indicating that it is unlikely to participate in catalysis. In agreement with this, mutation of Asp262 to asparagine (D262N, single letter amino acid code) in full length Pyst1 had no significant effect on the rate of p-NPP hydrolysis in the absence of ERK2. This contrasts with results obtained for both VHR and PTP1B, where substitution of this residue greatly decreases activity [19]. However, the D262N mutant of Pyst1 fails to undergo catalytic activation on addition of ERK2, indicating that this residue is only required for catalysis in the activated form of Pyst1 [16••]. In support of a role for catalytic activation in vivo, this mutant is also severely compromised in its ability to dephosphorylate ERK2 in mammalian cells. The simplest interpretation of these data is that Asp262 and its associated loop undergo closure over the active site of Pyst1 only when ERK2 is bound to the phosphatase, thus positioning the aspartate residue in an optimal conformation for catalysis. However, the precise nature of these conformational changes must await the determination of a structure of a co-complex of Pyst1 and ERK2.
Physiological roles for dual-specificity MKPs Despite the evidence in support of a role for these enzymes in regulating MAPKs and SAPKs in vivo, definitive proof is still lacking in mammalian systems. The mouse CL100 (MKP-1) gene has been disrupted and these animals develop normally and are fertile. Furthermore, cells cultured from these animals do not display any abnormalities in either MAPK activation or inactivation [20]. However, direct evidence of a role for these enzymes has come from genetic and biochemical studies in yeasts and Drosophila. The Msg5p enzyme of Saccharomyces cerevisiae is most similar in sequence to human CL100 (MKP-1), and was isolated as a suppressor of pheromone-induced G1 arrest, which is mediated by the Fus3p MAPK in haploid yeast cells [21]. Deletion of MSG5 results in increased activation of Fus3p, and Msg5p efficiently dephosphorylates both the threonine and tyrosine residues of Fus3p in vitro. The observation that MSG5 is pheromone inducible and that this induction is mediated by FUS3 indicates that this phosphatase is involved in negative feedback control of MAPK signalling. More recently, two further dual specificity MKPs have been identified in yeasts: CPP1, a gene which is highly related to MSG5, has been isolated from the human opportunistic pathogen Candida albicans and regulates a MAPK pathway involved in the yeast to hyphal transition [22]; the pmp1+ gene in Schizosaccharomyces pombe encodes a protein that is closely related to Msg5p and dephosphorylates the fission yeast MAPK Pmk1p in vitro and in vivo [23]. Pmk1p functions co-ordinately with a protein kinase C pathway and is implicated in the maintenance of cell integrity. This suggests that pmp1+, like MSG5, may be involved in feedback control of MAP kinase activity. It is not yet clear,
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Figure 2
Figure 3
(a)
(b) MEKK?
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Ssk2p/ssk22p
hep
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PTP-ER Eye development
Current Opinion in Cell Biology
MAPK signalling pathways in Drosophila are regulated by both dual specificity and PTPases. (a) During embryogenesis, the Drosophila JNK pathway is activated in the cells at the leading edge of the epidermis. Djun is then activated and is involved in mediating expression of the dual specificity MKP encoded by puc, and also dpp (TGFβ). Puc then inactivates bsk (JNK) thus regulating its own expression [26••]. Dpp is involved in regulating cellular events including those required for dorsal closure. (b) The Drosophila MAPK pathway which lies downstream of Ras, and contains Raf and the rolled (rl) MAPK, acts as a binary switch during eye development. Signalling triggers the differentiation of only one of five identical cells as an R7 neuron, whereas the lack of signal transmission in the other four cells causes them to adopt the non-neuronal cone cell fate. PTPER has now been identified as a tyrosine-specific MKP which inactivates rl and plays a physiological role in regulating this signalling pathway [40••].
Both tyrosine-specific and dual specificity MKPs coordinately regulate a MAPK signalling pathway in S. cerevisiae. (a) The Hog1p osmoregulatory MAPK in budding yeast is regulated by two tyrosine specific phosphatases encoded by PTP2 and PTP3 [27,28]. Of these, Ptp2p is the most critical. (b) Ptp3p also plays a role in regulating the pheromone-responsive MAPK Fus3p where it acts in concert with the inducible dual-specificity MKP encoded by MSG5 to regulate the adaptive response to pheromone [29]. Positive interactions are indicated by arrows while negative regulation is indicated by bars. Different line weights indicate potency of activities. Dual-specificity MKPs are boxed, whereas tyrosine-specific MKPs are underlined.
able to inactivate JNK. Interestingly, both loss and overexpression of puc have adverse effects on dorsal closure, indicating that JNK activity must be tightly controlled within critical limits for normal development to occur. Finally, the expression of puc is dependent on the activity of either bsk or its upstream activator, indicating that puc functions as part of a negative feedback control (Figure 2a).
Tyrosine-specific phosphatases also regulate MAPK signalling however, whether either Pm1p protein levels or activity are modulated in response to stimuli that activate Pmk1p. Perhaps the most informative experiments providing direct proof of a physiological role for a dual-specificity phosphatase in the regulation of MAP kinase activity in higher eukaryotes have come from studies in Drosophila. Basket (bsk), the Drosophila homologue of the mammalian JNK (or SAPK1) plays a role in the process of dorsal closure during embryogenesis [24]. Dorsal closure is a process in which lateral epithelial cells undergo morphological changes and migrate to cover the dorsal region of the embryo. Ring and Martinez-Ariaz [25] identified mutations in the puckered (puc) gene as affecting this process and puc was subsequently found to encode a CL100-like dual specificity phosphatase [26••]. Biochemical assays showed that extracts from wild type but not puc mutant embryos were
A role for PTPases in regulating MAPK first came from genetic and biochemical studies of the osmoregulatory MAPK pathways in yeasts. In S. cerevisiae, the osmoticstress-responsive Hog1p MAPK is co-ordinately regulated by two PTPases encoded by PTP2 and PTP3 [27,28]. One unexpected twist in this story came with the finding that PTP3, together with the gene encoding the dual specificity phosphatase MSG5, also plays a role in the regulation of the pheromone-responsive Fus3p MAPK [29]. This was the first indication that a single MAP kinase could be regulated by more than one class of protein phosphatase (Figure 3). In S. pombe, the functional equivalent of the Hog1p MAPK Sty1/Spc1 is also regulated by two PTPases Pyp1 and Pyp2 [30,31]. Furthermore, Pyp2 levels are increased by activation of Sty1/Spc1. Thus, like the inducible dual specificity MKPs, these enzymes are implicated in feedback control of MAPK activity.
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Given the high level of conservation of MAPK pathways from yeasts to man, it would be surprising if mammalian equivalents of these tyrosine-specific MKPs did not exist. Recent work has now led to the discovery of three gene families of closely related PTPases which regulate MAPK signalling in mammalian cells. STEP (striatal enriched phosphatase) and PTP-SL (STEP-like phosphatase), PTPases which are expressed in neuronal cells, were found to physically associate with ERK1 and ERK2 [32•]. On binding to ERKs, two distinct events occurred: firstly, the noncatalytic amino termini of both PTP-SL and STEP were phosphorylated; secondly, the MAPK was dephosphorylated and inactivated, specifically on the tyrosine residue within the regulatory T–E–Y motif. A 16-aminoacid motif within the noncatalytic amino-terminal domain of both PTPases was identified as being critical for ERK binding and dephosphorylation and was termed the kinase interaction motif (KIM). Expression of PTP-SL was also able to suppress ERK activation in response to EGF in Cos-7 cells [32•]. Similar results were obtained for the phosphatase PTPBR7, an enzyme which is closely related to PTP-SL and STEP [33]. Interestingly, both cytosolic and transmembrane isoforms of PTP-SL were able to downregulate ERK2 activity indicating that these enzymes might act on distinct pools of ERK2 within the cell.
What physiological role do these tyrosine-specific enzymes play? HePTP is able to suppress T cell receptor mediated activation and may modulate cytokine production [34,35•,36•], and the neuronal PTPase PC12-PTP1 (a rat homologue of PTPBR7) is inducible by NGF, indicating that it may play a role in modulating MAPK activity in response to growth and differentiation factors [39]. However, direct in vivo evidence for a physiological role of a PTP in regulating ERK/MAPK in higher eukaryotes has again come from genetic studies of MAPK signalling in Drosphila.
The related lymphoid-specific PTPases, haemopoietic PTP (HePTP) and leukocyte PTP (LC-PTP), were found to block antigen-dependent T-lymphocyte activation [34,35•,36•], and cellular targets of HePTP/LC-PTP were identified as ERK1 and ERK2. Like PTP-SL and STEP, HePTP binds to ERK2 through its noncatalytic amino-terminal domain, which also contains a KIM motif. HePTP was also able to bind p38 MAPK (SAPK2) but was unable either to form complexes with JNK (SAPK1), or to inhibit JNK activity [35•]. Similar results have been reported for PTP-SL [37], reinforcing the notion that, just as for the dual specificity MKPs, substrate selectivity is determined by specific protein–protein interactions.
Conclusions and future perspectives
Like both PTP-SL and STEP, the amino-terminal domain of HePTP is also phosphorylated by ERKs. Two sites of modification, Thr45 and Ser72, have been identified [35•]. Interestingly, mutation of these residues to alanine augments the activity of HePTP, indicating that phosphorylation might play a role in dissociating ERK and HePTP [35•]. This idea is lent further support by the recent finding that Ser23 in HePTP, which lies within the minimal KIM motif, is phosphorylated by cAMP-dependent protein kinase (PKA) in vitro and in vivo [38•]. Furthermore, modification of this residue reduces the binding of ERK2 to HePTP and this dissociation is associated with increased ERK2 activity. Thus, it appears that phosphorylation of HePTP by both MAPKs and PKA may modify the ability of the phosphatase to bind to its substrate. In the latter case, this may be an important point of cross-talk between the MAPK and cAMP signalling pathways.
The Drosophila rolled (rl) MAPK gene, which encodes a protein most similar to mammalian ERK1 and 2, is implicated in signalling downstream of multiple receptor tyrosine kinases. This MAPK pathway, which lies downstream of Ras1, acts as a binary switch to trigger cell differentiation during eye development. Karim and Rubin [40••] carried out a genetic screen for mutations which act negatively downstream of Ras1 and isolated a PTP, protein tyrosine phosphatase-ERK/enhancer of Ras1 (PTP-ER). PTP-ER targets rl and is most similar in amino acid sequence to PTP-SL, STEP and HePTP. Furthermore, a KIM motif is located within the amino-terminal noncatalytic domain of the protein, and PTP-ER binds to and inactivates MAPK both in vitro and in vivo (Figure 2b).
A large number of mammalian MKPs have now been identified. These include both dual specificity and tyrosine-specific enzymes (Table 1). More may yet be discovered. The dual specificity phosphatase VHR, which lacks the amino-terminal domain found in other dual specificity MKPs, has recently been reported to target ERK2 [41] and there is evidence indicating that serine/threonine-specific phosphatases may also regulate MAPKs [42,43]. Specific protein–protein interactions mediated by the noncatalytic amino-terminal domains of both classes of MKPs are critical for substrate specificity. In the case of both Pyst1 and PTP-SL, these are sufficiently robust to anchor ERK2 protein in the cytoplasm of mammalian cells [37,44]. Interestingly, although there is no discernable amino acid homology between the MAPK-binding domains of mammalian dual specificity and tyrosine-specific MKPs, recent evidence suggests that they may recognise their MAPK targets in a similar way. The sevenmaker mutant of ERK2 (D321N), a gain-of-function mutant first identified in Drosophila [45], is known to be resistant to inactivation by dual specificity MKPs [46,47]. In the case of Pyst1 (MKP-3), ERK2 (D321N) is unable to bind the amino-terminal domain of the phosphatase and cause catalytic activation [20]. Very recently, it has been shown that both human HePTP [36•] and Drosophila PTPER [40••] are also unable to form complexes with, or inactivate, this ERK2 mutant. It will be interesting to determine the relative affinities of these interactions,
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particularly where a dual specificity MKP and a tyrosinespecific enzyme target the same MAPK isoform. Finally, MAPK modules, in common with other signalling pathways are regulated by organisation into higher order complexes [3•]. These complexes, which include both upstream activators and also noncatalytic proteins such as scaffolds and inhibitory proteins, will determine where and when a particular pathway is active. We will need to understand how the MKPs are integrated into these signalling assemblies before we can fully understand how these enzymes participate in the overall control of these key pathways.
Update Since the submission of this review, two important papers have been published that are particularly relevant to this article [48•,49•].
Acknowledgements The author acknowledges the support of the Imperial Cancer Research Fund and would also like to thank David Slack and Ole-Morten Seternes for critical reading of the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Robinson MJ, Cobb MH: Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 1997, 9:180-186.
2.
Cohen P: The search for physiological substrates of the MAP and SAP kinases in mammalian cells. Trends Cell Biol 1997, 7:353-361.
3. •
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48. Brondello J-M, Pouyssegur J, McKenzie FR: Reduced MAP kinase • phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 1999, 286:2514-2517. This paper shows that the inducible nuclear MKP-1(CL100) phosphatase is a labile protein and is targeted for degradation by the ubiquitin-directed proteasome complex. Furthermore, phosphorylation of MKP-1 on two carboxyterminal serine residues by p42MAPK or p44MAPK led to stabilisation of the protein. These results reveal a new facet of a complex control mechanism which is designed to prevent the undesirable effects of long-term activation of these key mitogenic signalling pathways. 49. Zhan X-L, Guan KL: A specific protein–protein interaction accounts • for the in vivo substrate selectivity of Ptp3 towards the Fus3 MAP kinase. Genes Dev 1999, 13:2811-2827. Tyrosine specific MKPs are found in both S. cerevisiae and mammalian cells. However, the kinase interaction motif (KIM) identified in the non-catalytic amino terminus of the mammalian enzymes, which is responsible for MAPK binding, is not present in yeast phosphatases such as Ptp3p which targets the Fus3p MAP kinase. Here the authors demonstrate that Ptp3p does bind to its substrate Fus3p via its non-catalytic amino-terminal domain. However, the sequence motif responsible is more similar to those identified in the noncatalytic amino terminal domains of the mammalian dual-specificity MKPs. This paper reinforces the concept that the specificity of MAP kinase inactivation in vivo is governed by specific protein–protein interactions with the phiosphatase catalytic domain.