ReviewProtein phosphatases and the regulation of mitogen-activated protein kinase signalling
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 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 tyrosine-specific 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.
Section snippets
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 amino-terminal 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
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
Substrate binding causes catalytic activation of dual-specificity MKPs
The dephosphorylation of ERK2 MAPK by Pyst1 (MKP-3) 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 amino-terminal 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
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
Tyrosine-specific phosphatases also regulate MAPK signalling
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 osmotic-stress-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
Conclusions and future perspectives
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
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
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