Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling

We dedicate this manuscript to the memory of Véronique Volmat tragically deceased on April 22, 2002.
https://doi.org/10.1016/S0006-2952(02)01135-8Get rights and content

Abstract

Extracellular signals transduced via receptor tyrosine kinases, G-protein-coupled receptors or integrins activate Ras, a key switch in cellular signalling. Although Ras can activate multiple downstream effectors (PI3K, Ral …) one of the major activated pathway is a conserved sequential protein kinase cascade referred to as the mitogen activated protein (MAP) kinase module: Raf>MEK>ERK. The fidelity of signalling among protein kinases and the spatio-temporal activation are certainly key determinants for generating precise biological responses. The fidelity is ensured by scaffold proteins, a sort of protein kinase “insulators” and/or specific docking sites among the members of the signalling cascade. These docking sites are found in upstream and downstream regulators and MAPK substrates [Nat Cell Biol 2 2000 110]. The duration and the intensity of the response are in part controlled by the compartmentalisation of the signalling molecules. Growth factors promote nuclear accumulation and persistent activation of ERK (p42/p44 MAP kinases) during the entire G1 period with an extinction during S-phase. These features are exquisitely well controlled by (i) the temporal induction of the MAP kinase phosphatases, MKP1-3, and (ii) the compartmentalisation of the signalling molecules. We have shown that MKP1-2 induction is strictly controlled by the activation of the MAP kinase module providing evidence for an autoregulatory mechanism. This negative regulatory loop was further enhanced by the capacity of ERK to phosphorylate MKP1 and 2. This action reduced the degradation rate of these MKPs through the ubiquitin–proteasomal system [Science 286 1999 2514].

Whereas the two upstream kinases of the module, Raf and MEK remained cytoplasmic, ERK anchored to MEK in the cytoplasm of resting cells, rapidly translocated to the nucleus upon mitogenic stimulation. This process was rapid, reversible, and controlled by the strict activation of the MAPK cascade. Prevention of this nuclear translocation, by overexpression of a cytoplasmic ERK-docking molecule (inactive MKP3) prevented growth factor-stimulated DNA replication [EMBO J 18 1999 664]. Following long term stimulation, ERK progressively accumulated in the nucleus in an inactive form. This nuclear retention relied on the neosynthesis of short-lived nuclear anchoring proteins. Nuclear inactivation and sequestration was likely to be controlled by MAP kinase phosphatases 1 and 2. Therefore we propose that the nucleus represents a site for ERK action, sequestration and signal termination [J Cell Sci 114 2001 3433].

In addition, with the generation of mice invalidated for each of the ERK isoforms, we will illustrate that besides controlling cell proliferation the ERK cascade also controls cell differentiation and cell behaviour [Science 286 1999 1374].

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.

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