Synonyms

Mapk4 (Erk4); Mapk6 (Erk3)

Historical Background

Extracellular signal-regulated kinase 3 (Erk3) and Erk4 are atypical members of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases. The Erk3 and Erk4 genes were originally identified in 1991 and 1992, by homology cloning with probes derived from the MAP kinase Erk1 (Boulton et al. 1991; Gonzalez et al. 1992). In human, Erk3 is encoded by the MAPK6 gene located on chromosome 15q21.2. The MAPK4 gene present on chromosome 18q21.1 encodes Erk4. The high sequence identity of Erk3 and Erk4 proteins and the similar organization of their genes indicate that the two proteins are true paralogs. It is noteworthy that MAPK6 and MAPK4 are the only MAP kinase genes that are restricted to the vertebrate lineage.

Structure of Erk3 and Erk4

Erk3 and Erk4 are related protein kinases of 100 and 70 kDa, respectively, that define a distinct subfamily of MAP kinases (Coulombe and Meloche 2007). They are characterized by the presence of a catalytic kinase domain at the N-terminal end, which is 73% amino acid identical, and a long C-terminal extension. Although highly conserved in vertebrate evolution, the function of the C-terminal domain remains to be defined. By contrast to classical MAP kinases like Erk1/Erk2, which are activated by dual phosphorylation of the threonine and tyrosine residues in the conserved Thr-Xxx-Tyr motif of the activation loop, Erk3 and Erk4 contain a single phospho-acceptor site (Ser-Glu-Gly sequence) in their activation loop. In addition, they bear the sequence Ser-Pro-Arg instead of Ala-Pro-Glu in kinase subdomain VIII, and are the only protein kinases in the human genome to have an arginine residue at this position. The impact of these structural features on the regulation of Erk3 and Erk4 is unknown.

Expression of Erk3 and Erk4

Erk3 is expressed ubiquitously in adult mammalian tissues, whereas Erk4 shows a more restricted expression profile. The level of expression of the two kinases varies considerably between tissues, but the highest expression is found in the brain (Boulton et al. 1991; Kant et al. 2006; Turgeon et al. 2000). In the mouse embryo, they share a similar temporal pattern of regulation with a peak of expression at embryonic day 11, coincident with the time of early organogenesis (Rousseau et al. 2010). In vitro studies have shown that Erk3 mRNA is upregulated upon differentiation of P19 embryonal carcinoma cells to the neuronal or muscle lineage (Boulton et al. 1991).

At the subcellular level, Erk3 and Erk4 exhibit distinct localization. Erk3 is found in both the cytoplasm and the nucleus of a variety of cell types, whereas Erk4 is localized mainly in the cytoplasmic compartment (Aberg et al. 2006; Julien et al. 2003; Kant et al. 2006). The cytoplasmic localization of Erk3 and Erk4 requires an active nuclear export by a Crm1-dependent mechanism. In contrast to classical MAP kinases, the cellular distribution of Erk3 and Erk4 does not change in response to common mitogenic or stress stimuli. However, Erk3 localization is regulated through its interaction with MAP kinase-activated protein kinase 5 (MK5) (Schumacher et al. 2004; Seternes et al. 2004). MK5 is a member of the MAP kinase-activated protein kinase family of protein kinases that lie downstream of MAP kinase signaling cascades. The formation of a complex between Erk3 and MK5 results in the nuclear to cytoplasmic redistribution of both proteins. The kinase activity of either protein is dispensable for this relocalization. In addition to its effect on localization, MK5 also affect endogenous Erk3 expression. Embryonic fibroblasts prepared from MK5-deficient mice or HeLa cells transfected with a si-RNA targeting MK5 exhibit a marked reduction of Erk3 protein level (Seternes et al. 2004).

Interestingly, Erk3 and Erk4 proteins display different stability. Whereas Erk4 is a relatively stable protein, Erk3 was shown to be a highly unstable protein in proliferating cells, with a half-life of about 30 min (Aberg et al. 2006; Coulombe et al. 2003; Kant et al. 2006). Erk3 is constitutively degraded by the ubiquitin-proteasome pathway, and two regions in the N-terminal lobe of the kinase domain are both necessary and sufficient to target Erk3 for proteolysis (Coulombe et al. 2003). Thus, Erk3 biological activity is regulated at the level of cellular abundance. The short half-life of Erk3 has a physiological significance since the protein is stabilized and accumulates to high levels in the course of cellular differentiation and during mitosis. Indeed, Erk3 stability increases with time during the neurogenic and myogenic differentiation of PC12 and C2C12 cells, respectively, leading to protein accumulation (Coulombe et al. 2003). Upregulation of Erk3 during muscle differentiation is concomitant to accumulation of the cell cycle inhibitor p21Cip1 and cell cycle exit. Recent findings have shown that phosphorylation of four residues located in the extreme C-terminal extension stabilizes Erk3 protein, leading to its accumulation in mitosis (Tanguay et al. 2010).

Regulation of Erk3 and Erk4 Activity and Substrates

Little is known about the regulation of Erk3 and Erk4 biological activity. In intact cells, Erk3 and Erk4 are phosphorylated on Ser189 and Ser186, respectively, in the Ser-Glu-Gly motif of their activation loop (Cheng et al. 1996; Coulombe et al. 2003; Deleris et al. 2008; Perander et al. 2008). Unlike classical MAP kinases, this phosphorylation event is detected in resting cells and is not modulated by common mitogenic or stress stimuli.

Classical MAP kinases like Erk1 are multifunctional kinases that phosphorylate a vast array of substrates. However, Erk3 and Erk4 do not phosphorylate generic MAP kinase substrates such as myelin basic protein, microtubule-associated protein-2, c-Jun, or Elk-1 (Cheng et al. 1996), suggesting that they have a more restricted substrate specificity. The identification of MK5 as specific interaction partner and substrate of both Erk3 and Erk4 was an important step toward a better characterization of these kinases. Binding of Erk3 or Erk4 to MK5 results in the phosphorylation of MK5 on Thr182, and its resulting enzymatic activation (Aberg et al. 2006; Kant et al. 2006; Schumacher et al. 2004; Seternes et al. 2004). While the catalytic activity of Erk4 is required to activate MK5, its activation by Erk3 appears to depend both on the kinase activity of Erk3 as well as on a cytoplasmic scaffolding role of Erk3 with subsequent MK5 autophosphorylation. Importantly, Erk3 and Erk4 are bonafide physiological regulators of MK5 activity. The endogenous activity of MK5 is partially reduced in cells deprived in either Erk3 or Erk4, and the combined knockdown of the two kinases results in a greater reduction of MK5 activity (Aberg et al. 2006; Seternes et al. 2004).

Recently, a more complex interaction between Erk3/Erk4 and MK5 has been proposed (Deleris et al. 2008; Perander et al. 2008). According to these studies, the interaction between Erk3/Erk4 and MK5 recruits and/or contributes to the activation of Erk3/Erk4 activation loop kinase, resulting in the enhancement of Erk3/Erk4 phosphorylation on Ser189/Ser186. This, in turn, leads to the stabilization of Erk3/Erk4-MK5 complexes, the full enzymatic activation of Erk3/Erk4, and the phosphorylation of MK5 on activation loop Thr182. Activated MK5 reciprocally phosphorylates Erk3 and Erk4 on unidentified sites located outside the activation loop. The finding that the interaction between Erk3/Erk4 and MK5 requires activation loop phosphorylation of Erk3/Erk4 indicates that the mechanism by which these atypical MAP kinases bind to effector kinases is distinct to that of classical MAP kinases. Indeed, Erk1/Erk2 and p38 interact with downstream MAP kinase-activated protein kinases through the common docking (CD) domain, a cluster of negatively charged amino acids located near the kinase domain of MAP kinases. The conserved CD domain within Erk3 and Erk4 is dispensable for MK5 interaction.

Physiological Functions of Erk3 and Erk4

Much remain to be learned about the cellular functions of Erk3 and Erk4. However, in vitro studies in different model cell lines and in vivo analysis of mice deficient for Erk3 or Erk4 have started to shed light on the potential roles of these kinases. All these studies suggest that Erk3 and Erk4 are likely to be involved in the control of cell proliferation and differentiation. Erk3 expression is upregulated during differentiation of P19 and PC12 cells into neurons and of C2C12 cells into myotubes, in association with proliferation arrest (Boulton et al. 1991; Coulombe et al. 2003). It has been reported that the overexpression of stable forms of Erk3 in fibroblasts inhibits S-phase entry (Coulombe et al. 2003). Moreover, Erk3 was shown to interact with the cell cycle regulatory proteins cyclin D3, cell-division cycle 14A (Cdc14A) and Cdc14B through its C-terminal extension (Hansen et al. 2008; Sun et al. 2006; Tanguay et al. 2010). The phosphorylation of Erk3 also varies during the cell cycle. Erk3 is phosphorylated in the C-terminal extension during entry into mitosis and dephosphorylated at the M- to G1-phase transition. Cdc14A and Cdc14B phosphatases reverse the C-terminal phosphorylation of Erk3, and the mitotic kinase cyclin B1/cyclin-dependent kinase 1 (Cdk1) is most likely responsible for the phosphorylation of these sites. A potential role of Erk3 in insulin secretion has also been reported in another study (Anhe et al. 2006). Prolactin treatment of isolated rat pancreatic islets was found to increase Erk3 expression. This hormone is involved in the adaptive response of pancreatic β-cells toward peripheral insulin resistance during pregnancy. Silencing of Erk3 in isolated pancreatic islets prevents glucose-stimulated insulin secretion.

The study of mice deficient for Erk3, Erk4, and both Erk3 and Erk4 has provided important clues about their physiological roles. Loss of Erk3 leads to intrauterine growth restriction, delayed lung maturation associated with decreased sacculation and defective type II pneumocyte differentiation, and neonatal lethality (Klinger et al. 2009). Whereas the lung maturation defect can be overcome by in utero glucocorticoid administration, the newborn mice cannot be rescued from neonatal death. This indicates that additional physiological alterations contribute to the neonatal lethality. Erk3-deficient mice have reduced levels of insulin-like growth factor 2 (IGF-2) in the serum, suggesting that Erk3 might be a regulator of IGF-2 levels. Erk4-deficient mice are viable, develop normally, and show no gross physiological anomalies (Rousseau et al. 2010). Interestingly, behavioral analysis revealed that Erk4 mutant mice manifest depression-like behavior. In these mice, the loss of Erk4 is not compensated by increased activity or level of Erk3. Also, additional deletion of Erk4 in Erk3-deficient mice does not aggravate the fetal growth restriction and pulmonary immaturity phenotypes. This indicates that Erk3 and Erk4 protein kinases have acquired specific nonredundant functions through evolutionary diversification.

To date, MK5 remains the only identified substrate of Erk3 and Erk4, but the function of MK5 is still elusive. Of note, a recent study reported that mice deficient for MK5 display enhanced skin carcinogenesis after dimethylbenzanthracene (mutagen causing activating Ras mutations) application and compromised senescence induction (Sun et al. 2007). It remains to be determined whether Erk3 and Erk4 play a role in Ras-induced senescence and tumor suppression. The identification of additional substrates of Erk3 and Erk4 will help to better characterize the biological functions of these kinases and the signaling pathways in which they are involved.

Summary

Erk3 and Erk4 were among the first MAP kinases to be identified in the early 1990s. However, the characterization of these protein kinases has progressed much slower than that of classical MAP kinase family members such as Erk1/Erk2. Much still remains to be learned about their regulation, the identity, and spectrum of their substrates, their physiological roles, and their putative involvement in human diseases.