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The regulation of mitogen and stress-activated protein kinase signalling in mammalian cells
T ELSEVIER
Toxicology
148 (2000) 7-80
Invited Speakers’ Abstracts
The Regulation
of Mitogen
and Stress-Activated
Stephen M. Keyse ICRF Molecular Pharmacology DDl 9SY, Scotland, UK
Protein Kinase Signalling
in Mammalian
Cells
Unit, Biomedical Research Centre, Level 5 Ninewells Hospital, Dundee
Mitogen and stress-activated protein kinases (MAP/SAPKs) are key components of signal transduction pathways which amplify and integrate extracellular signals and mediate a diverse array of cellular responses. These include cellular proliferation, differentiation, development, inflammatory responses and apoptosis. It is now clear that both the duration and strength of MAP kinase signalling are key determinants of biological outcome. These parameters can be regulated at many points within the signalling pathway. However, it is now clear that a major point of regulation occurs at the level of the MAP kinase itself. MAPK activity reflects a balance between the activities of the upstream activating kinase and protein phosphatases. A family of dual-specificity MAPK phosphatases (MKPs) have been identified in mammalian cells. ‘These enzymes possess a catalytic domain, which is similar in sequence to the dual-specificity phosphatase VHl, and have an amino-terminal non-catalytic domain. which shares sequence and structural similarity to the cdc25 phosphatase. The prototypic mammalian MKP is CLlOO/MKP-1, which is inducible by many stimuli which cause activation of both mitogen and stress-activated protein kinases, including growth factors and oxidative stress. The latter observation suggests that these enzymes are involved in negative feedback regulation of MAP and SAP kinase signalling in mammalian cells. Recent work has now expanded this family of MKPs to at least ten members and it is clear that these proteins exhibit distinct patterns of subcellular localisation. Furthermore, certain MKPs typified by Pyst 1(MKP-3) are able to discriminate between different MAP and SAP kinase isoforms both in vitro and in vivo (Camps et al., 1998). The latter property is determined by the ability of the non-catalytic N-terminal domain of the MKPs to recognise and bind to substrate MAP kinases, an event which is accompanied by the catalytic activation of the MKPs in vitro (Dowd et al., 1998; Groom et al., 1996). We have recently determined the crystal structure of the catalytic domain of the mammalian Pystl (MKP-3) protein (Stewart et al., 1999). This reveals a shallow active site pocket, in which several of the key catalytic residues have misaligned sidechain conformations. We conclude that the striking enhancement of the catalytic activities seen on substrate binding reflects a conformational rearrangement of several catalytic sidechains within the MKP active site. Overall, these studies indicate that a complex interplay between upstream activators and multiple protein phosphatases is responsible for the regulation of MAP kinase activity. The activities, substrate specificities and subcellular localisation of these enzymes are likely to be key determinants of the biological outcome of signalling through these pathways in mammalian cells and tissues. 0300-483X’OO,$
- see front
PII: SO300-483X(00)00218-3
matter
0 2000 Elsevier
Science Ireland
Ltd. All rights
reserved
Abstracts
8
Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., Arkinstall, S., 1998. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280, 1262- 1265. Dowd, S., Sneddon, A.A., Keyse S.M., 1998. Isolation of the human genes encoding the Pystl and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases. J. Cell. Sci. 111, 3389-3399. Groom, L.A., Sneddon, A.A., Alessi, D.R., Dowd, S., Keyse, S.M. 1996. Differential regulation of MAP, SAP and RK/p38 kinases by Pystl, a novel cytosolic dual-specificity phosphatase EMBO J. 15: 3621-3622. Stewart, A.E., Dowd, S., Keyse, S.M., McDonald, N.Q., 1999. Crystal structure of the MAPK phosphatase Pystl catalytic domain and implications for regulated activation. Nature Struct. Biol. 6, 174-181.
Mechanism
and Activation
and Function of Protein Kinase B
Dario Alessi MRC Protein Phosphorylation 4HN
Unit, Department
of Biochemistry,
University of Dundee, Dundee DDl
Protein kinase B (PKB), also known as c-Akt, is activated rapidly when cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies closely downstream of phosphoinositide 3-kinase. Recent work has established that PKB is activated by its interaction with 3-phosphoinositide phospholipids. These lipids do not directly activate PKB, but instead recruit PKB to the membrane of cells and also induce a conformational change in PKB which makes it into a substrate for a novel kinase termed 3-phosphoinositide dependent protein kinase (PDKl). PDKl phosphorylates Thr308 in the kinase domain of PKB resulting in its partial activation. Once activated PKB plays a central role in insulin signalling, partly by phosphorylating and inactivating GSK3. PKB activation also protects cells from undergoing apoptosis. PDKl also phosphorylates and activates another insulin stimulated protein kinase termed the p7OS6 kinase, which plays a key role in the mechanism by which insulin activates protein synthesis. PDKl is likely to phosphorylate other protein kinases which belong to the same sub family of kinase as PKB and the p70 S6 kinase. The evidence supporting these finds will be presented at the meeting.
Signalling in Cell Survival and Cell Death
A.-N. Tony Kong, RongYu, Sandhya Mandlekar Center for Pharmaceutical Biotechnology, Department of Pharmaceutics and Pharmacodynamics, of Pharmacy, University of Illinois, Chicago, IL, USA
College
Cellular responses to xenobiotic-induced stress can signal proliferation, differentiation, homeostasis, apoptosis or necrosis. To better understand the underlying molecular mechanisms after exposure to xenobiotics or drugs, we investigated the signal transduction pathways, the mitogen-activated protein kinase (MAPK), and the ICE/Ced-3 protease (caspase) pathways, activated by different agents. The MAPKs, characterised as proline-directed serine/threonine kinases, are essential component of signalling pathways that convert various extracellular signals into intracellular responses through serial phosphorylation cascades (Karin, 1998). Once activated, MAPKs can phosphorylate many transcription factors, such as c-Myc, p62TCF/Elk-1, cJun, ATF-2, GADD153, and SAP-l, and ultimately leading to changes
Toxicology
148 (2000) 7-80
Invited Speakers’ Abstracts
The Regulation
of Mitogen
and Stress-Activated
Stephen M. Keyse ICRF Molecular Pharmacology DDl 9SY, Scotland, UK
Protein Kinase Signalling
in Mammalian
Cells
Unit, Biomedical Research Centre, Level 5 Ninewells Hospital, Dundee
Mitogen and stress-activated protein kinases (MAP/SAPKs) are key components of signal transduction pathways which amplify and integrate extracellular signals and mediate a diverse array of cellular responses. These include cellular proliferation, differentiation, development, inflammatory responses and apoptosis. It is now clear that both the duration and strength of MAP kinase signalling are key determinants of biological outcome. These parameters can be regulated at many points within the signalling pathway. However, it is now clear that a major point of regulation occurs at the level of the MAP kinase itself. MAPK activity reflects a balance between the activities of the upstream activating kinase and protein phosphatases. A family of dual-specificity MAPK phosphatases (MKPs) have been identified in mammalian cells. ‘These enzymes possess a catalytic domain, which is similar in sequence to the dual-specificity phosphatase VHl, and have an amino-terminal non-catalytic domain. which shares sequence and structural similarity to the cdc25 phosphatase. The prototypic mammalian MKP is CLlOO/MKP-1, which is inducible by many stimuli which cause activation of both mitogen and stress-activated protein kinases, including growth factors and oxidative stress. The latter observation suggests that these enzymes are involved in negative feedback regulation of MAP and SAP kinase signalling in mammalian cells. Recent work has now expanded this family of MKPs to at least ten members and it is clear that these proteins exhibit distinct patterns of subcellular localisation. Furthermore, certain MKPs typified by Pyst 1(MKP-3) are able to discriminate between different MAP and SAP kinase isoforms both in vitro and in vivo (Camps et al., 1998). The latter property is determined by the ability of the non-catalytic N-terminal domain of the MKPs to recognise and bind to substrate MAP kinases, an event which is accompanied by the catalytic activation of the MKPs in vitro (Dowd et al., 1998; Groom et al., 1996). We have recently determined the crystal structure of the catalytic domain of the mammalian Pystl (MKP-3) protein (Stewart et al., 1999). This reveals a shallow active site pocket, in which several of the key catalytic residues have misaligned sidechain conformations. We conclude that the striking enhancement of the catalytic activities seen on substrate binding reflects a conformational rearrangement of several catalytic sidechains within the MKP active site. Overall, these studies indicate that a complex interplay between upstream activators and multiple protein phosphatases is responsible for the regulation of MAP kinase activity. The activities, substrate specificities and subcellular localisation of these enzymes are likely to be key determinants of the biological outcome of signalling through these pathways in mammalian cells and tissues. 0300-483X’OO,$
- see front
PII: SO300-483X(00)00218-3
matter
0 2000 Elsevier
Science Ireland
Ltd. All rights
reserved
Abstracts
8
Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., Arkinstall, S., 1998. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280, 1262- 1265. Dowd, S., Sneddon, A.A., Keyse S.M., 1998. Isolation of the human genes encoding the Pystl and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases. J. Cell. Sci. 111, 3389-3399. Groom, L.A., Sneddon, A.A., Alessi, D.R., Dowd, S., Keyse, S.M. 1996. Differential regulation of MAP, SAP and RK/p38 kinases by Pystl, a novel cytosolic dual-specificity phosphatase EMBO J. 15: 3621-3622. Stewart, A.E., Dowd, S., Keyse, S.M., McDonald, N.Q., 1999. Crystal structure of the MAPK phosphatase Pystl catalytic domain and implications for regulated activation. Nature Struct. Biol. 6, 174-181.
Mechanism
and Activation
and Function of Protein Kinase B
Dario Alessi MRC Protein Phosphorylation 4HN
Unit, Department
of Biochemistry,
University of Dundee, Dundee DDl
Protein kinase B (PKB), also known as c-Akt, is activated rapidly when cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies closely downstream of phosphoinositide 3-kinase. Recent work has established that PKB is activated by its interaction with 3-phosphoinositide phospholipids. These lipids do not directly activate PKB, but instead recruit PKB to the membrane of cells and also induce a conformational change in PKB which makes it into a substrate for a novel kinase termed 3-phosphoinositide dependent protein kinase (PDKl). PDKl phosphorylates Thr308 in the kinase domain of PKB resulting in its partial activation. Once activated PKB plays a central role in insulin signalling, partly by phosphorylating and inactivating GSK3. PKB activation also protects cells from undergoing apoptosis. PDKl also phosphorylates and activates another insulin stimulated protein kinase termed the p7OS6 kinase, which plays a key role in the mechanism by which insulin activates protein synthesis. PDKl is likely to phosphorylate other protein kinases which belong to the same sub family of kinase as PKB and the p70 S6 kinase. The evidence supporting these finds will be presented at the meeting.
Signalling in Cell Survival and Cell Death
A.-N. Tony Kong, RongYu, Sandhya Mandlekar Center for Pharmaceutical Biotechnology, Department of Pharmaceutics and Pharmacodynamics, of Pharmacy, University of Illinois, Chicago, IL, USA
College
Cellular responses to xenobiotic-induced stress can signal proliferation, differentiation, homeostasis, apoptosis or necrosis. To better understand the underlying molecular mechanisms after exposure to xenobiotics or drugs, we investigated the signal transduction pathways, the mitogen-activated protein kinase (MAPK), and the ICE/Ced-3 protease (caspase) pathways, activated by different agents. The MAPKs, characterised as proline-directed serine/threonine kinases, are essential component of signalling pathways that convert various extracellular signals into intracellular responses through serial phosphorylation cascades (Karin, 1998). Once activated, MAPKs can phosphorylate many transcription factors, such as c-Myc, p62TCF/Elk-1, cJun, ATF-2, GADD153, and SAP-l, and ultimately leading to changes