Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling
Introduction
ERK (p42/p44 MAPK) constitutes a major signalling module conserved throughout evolution that is activated in mammalian cells via stimulation of receptor tyrosine kinases, G-protein coupled receptors and integrins [6]. These cell surface signals converge towards activation of the small G-protein Ras that recruits the serine/threonine kinase Raf to the membrane where it is fully activated by largely unknown mechanisms [7]. The signal is amplified via two downstream kinases, MEK and ERK that are uniquely activated since MEK is dually phosphorylated on two serine residues by Raf, and then ERK is dually phosphorylated on a tyrosine and threonine residue by MEK (sequence TEY). Amplification via this signalling cascade is such that it is estimated that activation of solely 5% of Ras molecules is sufficient to induce full activation of ERK [8].
Activated ERK phosphorylates numerous substrates on (S/T)P sites in all cellular compartments (review by [9]). Proper activation of the ERK pathway relative to the closely related JNK and p38 MAPK pathways and efficiency in transmitting activation occurs by two mechanisms. First, scaffolding proteins are expected to maintain in close vicinity the components of the ERK signalling cascade, and second, specific docking sites on substrates, activators and regulatory proteins maintain the specificity of activation. The first part of this presentation will unveil our current understanding of these scaffolding proteins and docking sites.
ERK activation is essential for cell growth [10] and provides an integrated response: it increases nucleotide synthesis, activates the transcription of many genes acting via transcription factors and chromatin phosphorylation, it stimulates protein synthesis via MNK1, and finally facilitates the formation of an active cyclinD–CDK4 complex, which is rate-limiting for cell growth (reviews by [11], [12]).
The specific role of the two ERK isoforms is not yet fully understood. First, both isoforms are ubiquitously expressed, second, they are highly similar (overall 75% identity at the amino acid level, and up to 90% identity when the N-terminal stretch is not taken into account) and third, in vitro both isoforms present the same substrate specificity. However, isoform-specific invalidation in mice provides contrasting results, first ERK1−/− mice are viable, fertile and of normal size [5]. In these animals ERK2 can compensate for most of the functions of ERK1, solely thymocyte terminal differentiation is impaired. On the contrary, ERK2 invalidation is lethal at early embryonic stages, day 6.5 (Sylvain Meloche, personal communication). In these embryos, ERK1 cannot compensate for loss of ERK2, thus specific functions of ERK2 remain to be discovered. Alternatively, ERK1 is not expressed in some cells or at such low levels compared to ERK2 that it cannot provide the strength of activation required for embryonic survival.
Considering the pleiotropic substrates and the ubiquitous expression of ERK, cell specific regulation must occur to ensure conduction of the appropriate signal. For example, expression of the ERK regulator PEA15 is restricted to only a few cell types such as terminally differentiated astrocytes. In these cells, expression of PEA15 attenuates ERK-dependent transcription and proliferation by binding ERK and re-addressing ERK signalling in the cytoplasm [13].
In a single cell, activation of the ERK pathway can lead to antagonistic fates, for example in PC12 cells both differentiation and cell proliferation require ERK activation (following NGF or EGF stimulation, respectively). In these cells EGF causes a transient activation of ERK, whereas NGF causes a sustained activation of ERK, thus the duration of ERK activation specifies signal identity [14]. Similarly, we have observed a correlation between the strength of mitogenic signalling in CCL39 cells and the duration of ERK stimulation. We have shown that non-mitogenic factors induce transient activation of ERK (less than 15 min) that does not lead to cell cycle entry whereas mitogens induce cell proliferation and long term stimulation of ERK (up to 6 hr) [15]. Similarly, it has been shown that very potent ERK activation protects cells from apoptosis induced by anchorage and serum removal [16], whereas moderate ERK activation is required to permit apoptosis induced by anchorage and serum removal [17].
Clearly the ERK pathway must be tightly controlled in its duration of activation and sub-cellular localisation to ensure proper outcome of integrated biological responses such as cell proliferation, differentiation and survival. The Section 3 of this presentation will describe the phosphatases that control the duration of stimulation, and Section 4 will present the regulation of ERK trafficking.
Section snippets
Scaffolding and docking sites
As indicated previously, several MAPK cascades delivering specific biological responses are present in a particular cell. There is a high degree of homology between MAPK modules, in their general organisation but also at the protein level with a high percentage of similarity in the primary sequence of the different MAPKs (60% between ERK1/2 and either JNK or p38 MAPK). Furthermore, the substrates of the three main MAPKs: ERK, JNK, and p38 MAPK display similar phosphorylation consensus motifs:
Regulation of ERK activation by phosphatases
Schematically, mitogenic stimulation elicits ERK activation in four phases. First, there is an initial burst of activation, second, there is a very rapid inactivation within minutes, third, there is a prolonged activation peaking from 2 to 4 hr poststimulation, and fourth, the activation gradually diminishes and ERK activity is reduced nearly to basal levels at the end of the G1 phase of the cell cycle. A burst of ERK activation has been described at the G2/M transition, but is beyond the scope
Trafficking
The sub-cellular localisation of ERK during serum stimulation in NIH-3T3 cells is presented in Fig. 1A. As described for other cell lines, ERK is accumulated in the cytoplasm of arrested NIH-3T3 cells (first panel). Within 10 min of stimulation (second panel), a major part of the pool of ERK translocates into the nucleus. At 1 hr after stimulation, ERK is distributed throughout the cell, slightly more in the nucleus than in the cytoplasm (third panel). Interestingly, maximal nuclear accumulation
Nuclear accumulation and inactivation
When activation of the ERK pathway is transient, ERK rapidly exits out of the nucleus [50], however during sustained activation, ERK accumulates in the nucleus as shown in Fig. 1A. The nuclear accumulation of ERK in the nucleus requires the ERK-dependent transcriptional induction of short-lived nuclear anchoring proteins [55]. The identity of these nuclear anchors remains elusive, however the use of anti-phospho-ERK antibodies provided new clues in understanding this nuclear accumulation of ERK.
Conclusions
ERK activation plays a major role in the integration of multiple biological responses. Hence, exquisite regulation of ERK activation is essential in conveying appropriate signals. The intensity, duration and sub-cellular localisation of ERK activation are well regulated. Scaffolding proteins and docking sites provide the means to avoid cross-activation between MAPK signalling pathways, and permit precise and even cell-specific sub-cellular localisation of ERKs.
We propose that the nucleus plays
Acknowledgements
We thank all the members of the laboratory for helpful discussions, and are particularly grateful to Dr. C. Brahimi-Horn for carefully reading the manuscript.
References (61)
- et al.
The Ras–Raf relationship: an unfinished puzzle
Adv. Enzyme Regul.
(2001) - et al.
Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation
J. Biol. Chem.
(1994) - et al.
Signal transduction through MAP kinase cascades
Adv. Cancer. Res.
(1998) - et al.
PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase
Dev. Cell
(2001) - et al.
Coordinate, biphasic activation of p44 mitogen activated protein kinase and S6 kinase by growth factors in hamster fibroblasts
J. Biol. Chem.
(1992) - et al.
Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals
Trends Biochem. Sci.
(1998) MAP kinases bite back
Dev. Cell
(2001)- et al.
Nuclear shuttling of yeast scaffold Ste5 is required for its recruitment to the plasma membrane and activation of the mating MAPK cascade [in process citation]
Cell
(1999) - et al.
KSR, a novel protein kinase required for Ras signal transduction
Cell
(1995) - et al.
C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1
Mol. Cell
(2001)
A conserved motif at the amino termini of MEKs might mediate high affinity interaction with the cognate MAPKs
Trends Biochem. Sci.
Two clusters of residues at the docking groove of mitogen-activated protein kinases differentially mediate their functional interaction with the tyrosine phosphatases PTP-SL and STEP
J. Biol. Chem.
Docking domains and substrate-specificity determination for MAP kinases
Trends Biochem. Sci.
A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission
J. Biol. Chem.
Docking sites on substrate proteins direct extracellular signal-regulated kinase to phosphorylate specific residues
J. Biol. Chem.
Cytoplasmic localization of MAP kinase kinase directed by its N-terminal, leucin-rich amino acid sequence, which acts as a nuclear export signal
J. Biol. Chem.
Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines
Curr. Biol.
PTP-ER, a novel tyrosine phosphatase, functions downstream of Ras1 to downregulate MAP kinase during Drosophila eye development
Mol. Cell
The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade
J. Biol. Chem.
Protein phosphatases, the regulation of mitogen-activated protein kinase signalling
Curr. Opin. Cell Biol.
Distinct binding determinants for ERK2/p38alpha and JNK map kinases mediate catalytic activation and substrate selectivity of map kinase phosphatase-1
J. Biol. Chem.
EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor
Curr. Biol.
Significance of nuclear relocalization of ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent fibroblasts
J. Biol. Chem.
Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation
Cell
Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells
J. Biol. Chem.
Spatiotemporal regulation of the p42/p44 MAPK pathway
Biol. Cell.
A conserved docking motif in MAP kinases common to substrates, activators and regulators
Nat. Cell. Biol.
Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation
Science
Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry
EMBO. J.
The nucleus, a site for signal termination by sequestration and inactivation of p42/p44 MAP kinases
J. Cell. Sci.
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